JARB Journal of Animal Reproduction and Biotehnology

OPEN ACCESS pISSN: 2671-4639
eISSN: 2671-4663

Article Search

Original Article

Article Original Article
Split Viewer

Journal of Animal Reproduction and Biotechnology 2020; 35(4): 307-314

Published online December 31, 2020

https://doi.org/10.12750/JARB.35.4.307

Copyright © The Korean Society of Animal Reproduction and Biotechnology.

Spermatozoa motility, viability, acrosome integrity, mitochondrial membrane potential and plasma membrane integrity in 0.25 mL and 0.5 mL straw after frozen-thawing in Hanwoo bull

Sung-Sik Kang , Ui-Hyung Kim , Myung-Suk Lee , Seok-Dong Lee and Sang-Rae Cho*

Hanwoo Research Institute, National Institute Animal Science (NIAS), Rural Development Administration (RDA), Pyeongchang 25340, Korea

Correspondence to: Sang-Rae Cho
E-mail: chosr@korea.kr
ORCID https://orcid.org/0000-0003-0209-6248

Received: October 20, 2020; Revised: November 17, 2020; Accepted: November 30, 2020

In the present study, we examined the effect of straw size on spermatozoa motility, viability, acrosome integrity, mitochondrial membrane potential, and plasma membrane integrity after freezing-thawing. Hanwoo semen was collected from three bulls and diluted with an animal protein-free extender, divided into two groups, namely, 10 million spermatozoa in 0.25 mL and 20 million spermatozoa in 0.5 mL straw, and cryopreserved. In Experiment 1, the motility and motility parameters of the frozen-thawed spermatozoa were evaluated. After freezing-thawing, the spermatozoa motility parameters fast progressive, straight line velocity, and average path velocity were compared between the 0.25 mL straw and 0.5 mL straw groups. They were 35.2 ± 1.0 and 32.3 ± 0.7%, 34.6 ± 0.7 and 31.8 ± 0.5 µm/s, 51.4 ± 1.3 and 47.1 ± 1.1 µm/s, 0.25 mL straw and 0.5 mL straw groups, respectively. In Experiment 2, the viability, acrosome membrane integrity, and mitochondrial membrane potential of the frozen-thawed spermatozoa were assessed. After freezing-thawing, the percentages of spermatozoa with live, intact acrosomes and high mitochondrial membrane potential were compared between the in 0.25 mL straw and 0.5 mL straw groups. They were 48.0 ± 2.6% and 35.6 ± 2.8% between the 0.25 mL straw and 0.5 mL straw groups. In Experiment 3, the plasma membrane integrity of frozen-thawed spermatozoa was compared. After freezing-thawing, the plasma membrane integrity was higher for the in 0.25 mL straw group than the 0.5 mL straw group. They were 62.0 ± 2.2 and 54.1 ± 1.3% between the 0.25 mL straw and 0.5 mL straw groups. In conclusion, our results suggest that freezing semen in 0.25 mL straw improves the relative motility, viability, and acrosomal, mitochondrial membrane potential, and plasma membrane integrity of Hanwoo bull spermatozoa.

Keywords: bull, freezing-thawing, spermatozoa, straw size

Natural mating with superior bulls and artificial insemination (AI) using semen collected from elite bulls have been globally applied in cattle breeding (Lima et al., 2009). However, natural mating may spread diseases borne by bulls. The adoption of AI in cattle reproduction mitigates the risk of disease transmission via bulls and lowers breeding costs (Valergakis et al., 2007). Elite bull semen may be transported to other states within the same country and to other countries (López-Gatius, 2012). The desired phenotypes and genes may be transferred to the offspring (Berry et al., 2020). In AI, semen is collected from elite bulls, diluted with suitable semen extenders, and stored in liquid or frozen solid form (Vishwanath and Shannon, 2000). Liquid semen is used in dairy cow reproduction with short (2-mo) short breeding seasons. In Ireland and New Zealand, it is adjusted according to pasture growth (Butler, 2014) and grass nutrient levels (Yang et al., 2018). However, liquid semen in extender can only provide conception rates comparable to those of frozen-thawed semen within the first 3 d (Vishwanath and Shannon, 2000). In contrast, frozen semen is semi-permanently cryopreserved in liquid nitrogen (LN2) and is generally used more often than liquid semen in AI (Layek et al., 2016). In general, semen collected from elite sires is diluted with semen extenders, filled in 0.25 mL or 0.5 mL straw, and cryopreserved in LN2 before use (Diskin, 2018). About 10-30 million spermatozoa are filled in 0.25 mL or 0.5 mL straw, distributed to farms, and used for AI worldwide (Stevenson et al., 2009). The 0.25 mL straw has half the volume of the 0.5 mL straw and more than two 0.25 mL straws can be preserved in the LN2 tank (Johnson et al., 1995). AI technicians and dairy and beef farm owners are often concerned that the low spermatozoa may reduce the conception rate. Nevertheless, there was no reduction in the conception rate after the introduction of 10-30 million spermatozoa for AI (Stevenson et al., 2009).

The Hanwoo Bull Center (Hanwoo Improvement Center, NH, Seosan, Korea) selected elite Hanwoo bulls and produced ~10 million frozen straws per Hanwoo bull lifespan. The Hanwoo Bull Center reported that it packed 18 million spermatozoa per 0.5 mL straw and distributed the frozen straws to all parts of Korea. Asia, Central and South America, and the United States produced 0.5 mL frozen straw. Canada and Europe commercially produced 0.25 mL frozen straw (Diskin, 2018). If 0.25 mL straw is substituted for 0.5 mL straw in semen freezing, the space in the LN2 cryopreservation tank can be doubled. Moreover, frozen straw production time and extender consumption can be reduced. However, the 0.25 mL straw has a larger exposure space than the 0.5 mL straw. Furthermore, the former is more sensitive than the latter to the external LN2 tank temperature and air currents. Therefore, short application time and minimum temperature change are required when 0.25 mL frozen straw is used in AI (Johnson et al., 1995). Despite these precautions, 0.25 mL straw has several advantages over 0.5 mL straw especially when the semen is diluted with tris-citric acid extender supplemented with 20% egg yolk (Anzar et al., 2011). For dairy bulls, frozen-thawed spermatozoa in 0.25 mL straw showed high motility and viability, low incidence of damaged acrosomal membranes (Senger et al., 1983), and high mitochondrial membrane potential compared to frozen-thawed spermatozoa in 0.5 mL straw (Anzar et al., 2011). Until recently, Hanwoo bull semen was only packed in 0.5 mL straw and to the best of our knowledge there were no reports comparing the properties of frozen-thawed spermatozoa in 0.25 mL and 0.5 mL straw.

In the present study, then, we investigated the effects of packing semen in 0.25 mL and 0.5 mL straw after freezing-thawing and compared their relative preservation efficacy. In Experiment 1, we compared the motility and motility parameters of frozen-thawed spermatozoa packed in 0.25 mL and 0.5 mL straw. To this end, we used a computer sperm analysis system. In Experiment 2, we examined the viability, acrosomal membrane integrity, and mitochondrial membrane potential of spermatozoa in 0.25 mL and 0.5 mL straw. For this purpose, we used fluorescent staining after freezing-thawing. In Experiment 3, we applied the hypoosmotic swelling test to study the plasma membrane integrity of spermatozoa in 0.25 mL and 0.5 mL straw after freezing-thawing.

Bull semen collection and cryopreservation

Bull semen was collected by an electro-ejaculator machine (Electro Jac 6; Neogen Corp., Lansing, MI, USA) between February and March 2020 at the Hanwoo Research Institute, NIAS, RDA, Pyeongchang, Korea. Three bulls aged 15 mo with mean weight 461.0 ± 47.8 kg were used for semen collection. Bulls A and B had two ejaculates each and bull C had one ejaculate. Semen volume, spermatozoa concentration and pH of raw semen were examined before dilution. Spermatozoa motility and motility parameters of raw semen were evaluated after dilution with semen extender and presented in Table 1. Spermatozoa motility was evaluated with a computer-assisted analytical system (CASA; sperm class analyzer; Microoptic SL, Barcelona, Spain). Over 90% of the motile spermatozoa sample was introduced for semen freezing. In brief, the semen was diluted with animal protein-free diluent (Optixcell; IMV Technologies, L’Aigle, France). The spermatozoa concentration was adjusted to 40 × 106 cells/mL and the dilution was cooled in a refrigerator at 4℃ for 3-4 h. The diluted semen was then introduced into 0.25 mL straw (10 × 106 spermatozoa/straw) and 0.5 mL straw (20 × 106 spermatozoa/straw). Spermatozoa density was identical for both the 0.25 mL and 0.5 mL straw. Straws were preserved by immersion for 14 min at 3 cm above the surface of LN2 in a styrofoam box. The straws were then dropped in LN2. The frozen straws were cryopreserved in LN2 tanks until spermatozoa motility and characteristics were evaluated.

Table 1 . The basic characteristics of raw semen, spermatozoa motility and motility parameters after dilution.

Raw semen informationMean value (5 replicates)
Volume (mL)3.8 ± 0.7
Sperm concentration (×106 cells/mL)828.5 ± 252.6
pH7.9 ± 0.2
Total motile (%)99.7 ± 0.1
Fast progressive (%)27.0 ± 3.7
Slow progressive (%)65.7 ± 4.3
Non-progressive (%)7.1 ± 1.2
Immotile (%)0.3 ± 0.1
VCL (μm/s)140.3 ± 1.1
VSL (μm/s)39.7 ± 3.2
VAP (μm/s)79.5 ± 5.5
LIN (%)28.6 ± 2.1
STR (%)50.0 ± 2.6
ALH (μm/s)4.0 ± 0.5
BCF (Hz)15.3 ± 0.7

Mean ± SE. Volume, sperm concentration and pH of raw semen were examined after semen collection. Spermatozoa motility and motility parameters were examined after dilution with semen extender. Values indicate mean of 5 replicates from each ejaculates..



Spermatozoa motility and motility parameters after freezing-thawing

Spermatozoa motility was examined as previously described (Yang et al., 2015; Kang et al., 2016), with slight modifications. In brief, frozen semen was immersed in water at 37.5℃ for 40 s and mixed in a 1.5 mL tube. Then 3 μL frozen-thawed semen was introduced to the chamber of a microscope slide (SC 20-01-04-B; Leja, Nieuw-Vennep, Netherlands). At least 800 spermatozoa in 4-5 fields per slide chamber were counted and spermatozoa motility and motility parameters were evaluated by CASA. Spermatozoa motility and the percentages of total motile, fast progressive, slow progressive, non-progressive, and immotile spermatozoa were evaluated. The other spermatozoa motility parameters assessed included curvilinear velocity (VCL, μm/s), straight line velocity (VSL, μm/s), average path velocity (VAP, μm/s), linearity (LIN = VSL/VCL, %), straightness (STR = VSL/VAP, %), amplitude of lateral head (ALH, μm/s), and flagellar beat cross frequency (BCF, Hz).

Spermatozoa viability, acrosomal membrane integrity, and mitochondrial membrane potential of after freezing-thawing

Spermatozoa characteristics were measured according to a previous report (Celeghini et al., 2007), with a modification. Frozen-thawed semen in 0.25 mL and 0.5 mL straw was transferred to a 1.5 mL tube and vortexed for 3 s. One hundred microliters frozen-thawed semen was diluted with 900 μL pre-warmed DPBS (-) at 37℃. One hundred microliters diluted semen was mixed with 150 μL of 5,5’,6,6’-tetrachloro-1,1’,3,3’tetraethylbenzimidazolylcarbocyanine iodide (JC-1; mitochondrial membrane potential detection kit; Cell Technology Inc., Danvers, MA, USA) working solution and incubated for 30 min at 37℃ in the dark. After incubation, 1 μL Hoechst 33342 (H1339; Molecular Probes, Eugene, OR, USA) stock solution was mixed with diluted semen and incubated for 10 min at 37℃ in the dark. Then 1 μL propidium iodide (PI; P-4172; Sigma-Aldrich Crop., St. Louis, MO, USA) stock solution and 1 μL fluorescein peanut agglutinin FITC conjugate (FITC-PNA; FL-1071; Vector Laboratories, Piedmont, Italy) were mixed with diluted semen and incubated for 8 min at 37℃ in the dark. Two microliters of 10% (v/v) formaldehyde was added to the stained semen mixture to impede spermatozoa movement (Harrison and Vickers, 1990). Five microliters stained semen was mounted on a slide glass and covered with a cover slip. More than 200 spermatozoa per microscope slide were counted at × 400 magnification under a fluorescent microscope (Eclipse Ti; Nikon, Tokyo, Japan). Live and dead spermatozoa, acrosomal membrane integrity, and mitochondrial membrane potential were assessed with a triple band filter (DAPI/FITC/TRITC; Nikon, Tokyo, Japan). The heads of live spermatozoa were stained with Hoechst 33342 (blue) while those of dead spermatozoa were stained with PI (red). High mitochondrial membrane potential was indicated by orange JC-1 staining of the spermatozoa midpiece. Low mitochondrial membrane potential was indicated by faint orange or no JC-1 staining of the spermatozoa midpiece. Damaged acrosomal membranes were stained with FITC-PNA (green) at the anterior spermatozoa head while intact acrosomal membranes were not stained at the anterior spermatozoa head.

Fluorescence preparation

One milligram Hoechst 33342 was diluted with 960 μL DPBS (-) and 40 μL dimethyl sulfoxide (DMSO) and cryopreserved at -20℃. The JC-1 stock solution contained 1 mg/mL DMSO in a mitochondrial membrane potential detection kit. To prepare the JC-1 working solution, 5 μL JC-1 stock solution at -20℃ and 500 μL DPBS (-) were mixed and cryopreserved at -20℃. PI stock solution was prepared by mixing 25 mg PI and 1 mL DMSO, diluting this mixture to 2 mg/mL with DPBS (-), and cryopreserving the dilution at -20℃. All fluorescent probe stock solutions except FITC-PNA were stored at -20℃ in the dark. The latter was preserved at 4℃. A 10% (v/v) formaldehyde working solution was prepared by mixing 2.9 mL of 35% (v/v) formaldehyde (Daejung Chemical & Metals Co., Gyeonggi-do, Korea) with 7.1 mL DPBS (-) and storing the dilution at 4℃.

Spermatozoa plasma membrane integrity after freezing-thawing

Spermatozoa plasma membrane integrity was determined as previously described (Kang et al., 2019). After freezing-thawing semen in 0.25 mL and 0.5 mL straw, the thawed semen was transferred to a 1.5 mL tube. Thirty microliters frozen-thawed semen was diluted with 300 μL hypoosmotic swelling test solution (Correa and Zavos, 1994) and incubated for 40 min at 37℃. Five microliters incubated semen was mounted on a glass microscope slide and covered with a cover slip. More than 200 spermatozoa per microscope slide were examined at × 400 magnification and scored as swelling or non-swelling. Swelling spermatozoa were considered to have intact plasma membranes.

Statistical analysis

Motility, motility parameters, viability, acrosomal membrane integrity, mitochondrial membrane potential, and plasma membrane integrity were analyzed by one-way ANOVA followed by Duncan’s test as a post hoc analysis. All analyses were performed in SAS v. 9.2 (SAS Institute, Cary, NC, USA). Spermatozoa viability, acrosomal membrane integrity, and mitochondrial membrane potential were calculated as %. Spermatozoa stained blue on head and orange on midpiece considered to have live, intact acrosomes and high mitochondrial membrane potential (LIAH). Spermatozoa stained blue on head and orange or colorless on midpiece considered to have live, intact acrosomes and low mitochondrial membrane potential (LIAL). Spermatozoa stained red on head considered to have dead, intact acrosomes and low mitochondrial membrane potential (DIAL). Spermatozoa stained red on postal head, green on arterial head, and colorless on midpiece considered to have dead, damaged acrosomes and low mitochondrial membrane potential (DDAL).

Experiment 1

To examine the effects of 0.25 and 0.5 mL straw, we compared frozen-thawed spermatozoa motility in 0.25 mL and 0.5 mL straw. Table 2 shows that the % of fast-progressive spermatozoa in the 0.25 mL straw group was significantly higher than that in the 0.5 mL straw group (35.2 ± 1.0 vs. 32.3 ± 0.7%, respectively; p < 0.05). The % of slow progressive in the 0.25 mL straw group was significantly lower than that in the 0.5 mL straw group (Table 2) (22.9 ± 0.7 vs. 26.0 ± 0.6%. respectively; p < 0.01). The % of VSL in the 0.25 mL straw group was significantly higher than that in 0.5 mL straw group (34.6 ± 0.7 vs. 31.8 ± 0.5%, respectively; p < 0.01). The % of VAP in the 0.25 mL straw group was significantly higher than that in 0.5 mL straw group (51.4 ± 1.3 vs. 47.1 ± 1.1%, respectively; p < 0.05).

Table 2 . Spermatozoa motility and motility parameters after freezing-thawing.

Straw size (replicates)

0.25 mL (25)0.5 mL (25)
Total motile (%)92.1 ± 1.388.7 ± 1.3
Fast progressive (%)35.2 ± 1.0a32.3 ± 0.7b
Slow progressive (%)34.0 ± 1.630.4 ± 1.7
Non-progressive (%)22.9 ± 0.7d26.0 ± 0.6c
Immotile (%)7.9 ± 1.311.3 ± 1.3
VCL (μm/s)85.5 ± 2.879.8 ± 2.4
VSL (μm/s)34.6 ± 0.7c31.8 ± 0.5d
VAP (μm/s)51.4 ± 1.3a47.1 ± 1.1b
LIN (%)41.0 ± 0.840.4 ± 0.8
STR (%)67.6 ± 0.868.0 ± 0.9
ALH (μm/s)3.1 ± 0.13.1 ± 0.1
BCF (Hz)15.3 ± 0.314.6 ± 0.1

a,bValues (mean ± SE) with different superscripts in the same row are significantly different (p < 0.05). c,dValues (mean ± SE) with different superscripts in the same row are significantly different (p < 0.01). Five straws of frozen thawed semen were used to evaluate sperm motility per ejaculation group. Values indicate mean of 25 replicates (5 replicates × 5 ejaculates)..



Experiment 2

Spermatozoa viability, acrosomal membrane integrity, and mitochondrial membrane potential after freezing-thawing were examined by the quadruple staining method. Table 3 shows that the % of LIAH in the 0.25 mL straw group was significantly higher than that in the 0.5 mL straw group (48.0 ± 2.6% vs. 35.6 ± 2.8%, respectively; p < 0.01). The % of LIAL in the 0.25 mL straw group was significantly lower than that in 0.5 mL straw group (8.4 ± 0.7 vs. 16.6 ± 1.8%, respectively; p < 0.001).

Table 3 . Viability, acrosomal membrane integrity, and mitochondrial membrane potential of spermatozoa after freezing-thawing.

Straw size (mL)ReplicatesLIAHLIALDIALDDAL
0.252548.0 ± 2.6c8.4 ± 0.7f23.1 ± 2.018.6 ± 1.6
0.52535.6 ± 2.8d16.6 ± 1.8e25.7 ± 2.120.4 ± 1.2

c,dValues (mean ± SE) with different superscripts in the same column are significantly different (p < 0.01). e,fValues (mean ± SE) with different superscripts in the same column are significantly different (p < 0.001). Values indicate mean of 25 replicates (5 replicates × 5 ejaculates). Spermatozoa with live intact acrosome and high mitochondrial membrane integrity, LIAH; spermatozoa with live intact acrosome and low mitochondrial membrane integrity, LIAL; spermatozoa with dead intact acrosome and low mitochondrial membrane integrity, DIAL; spermatozoa with dead damaged acrosome and low mitochondrial membrane integrity, DDAL; five straws of frozen-thawed semen were used to evaluate spermatozoa characteristics per ejaculation group..



Experiment 3

The differences in plasma membrane integrity between the 0.25 mL and 0.5 mL straw groups are shown in Table 4. The % of intact plasma membranes in the 0.25 mL straw group was significantly higher than that in the 0.5 mL straw (62.0 ± 2.2 vs. 54.1 ± 1.3%, respectively, p < 0.01).

Table 4 . Plasma membrane integrity of spermatozoa after freezing-thawing.

Straw size (mL)ReplicatesIntact plasma membrane (%)*
0.252562.0 ± 2.2c
0.52554.1 ± 1.3d

c,dValues (mean ± SE) with different superscripts in the same column are significantly different (p < 0.01). Asterisk indicates % intact plasma membrane spermatozoa. Five straws of frozen-thawed semen were used to evaluate intact plasma membrane spermatozoa per ejaculation group. Swollen spermatozoa were scored as spermatozoa with intact plasma membranes..


The sizes of the straws used to freeze-thaw bull semen vary with continent and country. Canada and Europe have adopted 0.25 mL straw while the United States and South America have adopted 0.5 mL straw. The optimal straw size and fertility rate are disputed among herdsman and technicians (Stevenson et al., 2009). In Korea, bull semen has been cryopreserved in 0.5 mL straw with semen extender based on tris-citric acid supplemented with 20% egg yolk. The latter has been used as a cryoprotectant in semen extender (Pace and Graham, 1974). However, animal proteins derived from egg yolk has disadvantages such as bacterial and mycoplasmal contamination (Bousseau et al., 1998). Recently, animal protein-free semen extenders have been investigated (Anzar et al., 2019). Here, we used an animal protein-free extender for Hanwoo bull semen. To the best of our knowledge, this work is the first to compare Hanwoo spermatozoa characteristics after freezing-thawing in 0.25 mL and 0.5 mL straw containing animal protein-free extender. We determined the relative effects of two different straw sizes on spermatozoa motility, viability, acrosomal membrane integrity, mitochondrial membrane potential, and plasma membrane integrity after freezing-thawing.

Spermatozoa motility and motility parameters have been used to predict fertility in livestock animals (Jepson et al., 2019). The percentages of fast progressive, VSL, and VAP for spermatozoa after thawing in 0.25 mL straw were significantly higher than those for spermatozoa after thawing in 0.5 mL straw (Table 2). These findings are consistent with a previous report showing that the fast progressive, VSL, and VAP of spermatozoa in 0.25 mL straw were higher than those of spermatozoa in 0.5 mL straw after 2 h thawing (Anzar et al., 2011). Enhanced fast progressive sperm motility in frozen-thawed bull semen increased its relative fertility (Morrell et al., 2017). The VSL and VAP were highly correlated with fertility in vitro (Kathiravan et al., 2008) and with fertility prediction in vivo (Farrell and Brockett, 1998). These earlier studies demonstrated that spermatozoa quality in 0.25 mL straw after freezing-thawing was superior to that of spermatozoa after freezing-thawing in 0.5 mL straw. Semen freezing in 0.25 mL straw augments post-AI fertility compared to that for semen freezing in 0.5 mL straw.

The proportion of spermatozoa with live, intact acrosome membranes, and high mitochondrial membrane potential (LIAH) was ~12% higher in 0.25 mL straw than it was in 0.5 mL straw (Table 3). Previous reports indicated higher LIAH for spermatozoa in 0.25 mL straw than those in 0.5 mL straw. An elevated proportion of LIAH may improve relative fertility (Anzar et al., 2011). Ansari et al. (2011) reported that the 0.25 mL straw group had 14% more viable spermatozoa than the 0.5 mL straw. The acrosome reaction is essential for fertilization. Cryopreservation and thawing induce the acrosome reaction in spermatozoa (Birck et al., 2010). The reduction of the acrosome reaction in spermatozoa during freezing and thawing is critical for increasing the relative proportion of spermatozoa with intact acrosomes (Nagata et al., 2019). Mitochondrial membrane potential is a predictor of in spermatozoa fertility potential in boars and bulls (Hu et al., 2017), stallions (Meyers et al., 2019), and humans (Kasai et al., 2002). The plasma membrane integrity of spermatozoa in 0.25 mL straw was 7.9% higher that of spermatozoa in 0.5 mL straw after freezing-thawing (Table 4). Elevated plasma membrane integrity of spermatozoa is an important indicator of bull fertility (Correa et al., 1997; Januskauskas et al., 2003). Hence, we propose that freezing bull semen in 0.25 mL straw increases spermatozoa motility, viability, acrosome integrity, mitochondrial membrane potential, and plasma membrane integrity.

In the present study, we assessed spermatozoa motility by CASA and spermatozoa characteristics by fluorescent staining and HOST. Reductions in spermatozoa quality by semen dilution, freezing, and thawing processes are inevitable in frozen semen fabrication (Chaveiro, 2006). As it has a large surface-to-volume, the 0.25 mL straw induces faster cooling and thawing than the 0.5 mL straw (Mocé et al., 2010). Cooling acceleration in 0.25 mL straw causes spermatozoa to pass through the fracture temperature and minimizes intracellular crystallization (Morris, 2006). However, the relatively improved motility, viability, acrosome membrane integrity, mitochondrial membrane potential, and plasma membrane integrity of the spermatozoa in 0.25 mL straw in vitro does not necessarily guarantee enhanced conception rates in vivo after AI. In fact, AI with 0.25 mL straw conferred only a 0.74% improvement in the expected fertility relative to AI using 0.5 mL straw (Stevenson et al., 2009). The use of 0.25 mL and 0.5 mL straw varies with country and continent and fertility rates vary with AI technician skills and straw handling (Diskin, 2018). The 0.25 mL straw is sensitive to exposure time, thawing conditions, and transportation after freezing-thawing (Stevenson et al., 2009). Future studies should evaluate the post-AI fertility rates using Hanwoo frozen-thawed spermatozoa in 0.25 mL straw. Moreover, the effects of freezing and thawing in 0.25 mL straw on spermatozoa characteristics should also be examined.

The present study demonstrated that semen freezing in 0.25 mL straw improved relative spermatozoa motility, viability, acrosome integrity, mitochondrial membrane potential, and plasma membrane integrity after freezing-thawing. The use of 0.25 mL straw doubles the number of straws that can be processed simultaneously in the LN2 tank compared to the 0.5 mL straws. Nevertheless, the in vivo post-AI fertility rates obtained with 0.25 mL straw should be investigated.

This study received support from the Cooperative Research Program for Agriculture Science & Technology Development project: “Development of improved technologies for preservation of Hanwoo bull semen and technology application”, PJ0143252020, RDA, Korea, and the 2020 Postdoctoral Fellowship Program of the Hanwoo Research Institute, NIAS, RDA, Korea.

We thank Mr. In-Rak Yoon, Mr. Woo-Heon Choi, Mr. Jong-Hwan Jang, Mr. Kyu-Myung Kim who care for bulls, supporting of semen collection and semen cryopreservation in Hanwoo Research Institute.


Conceptualization: Kang SS, Cho SR

Data curation: Kang SS

Formal analysis: Kang SS

Funding acquisition: Cho SR, Kim UH

Investigation: Kang SS, Lee MS, Lee SD

Methodology: Kang SS, Lee MS, Lee SD

Project administration: Cho SR

Resources: Cho SR

Software: Kang SS

Supervision: Cho SR

Validation: Kang SS, Cho SR, Kim UH

Visualization: Kang SS

Writing - original draft: Kang SS

Writing - review & editing: Kang SS

  1. Ansari MS, Rakha BA, Akhter S. 2011. Effect of straw size and thawing time on quality of cryopreserved buffalo (Bubalus bubalis) semen. Reprod. Biol. 11:49-54.
    Pubmed CrossRef
  2. Anzar M, Boswall L. 2011. Cryopreservation of bull semen shipped overnight and its effect on post-thaw sperm motility, plasma membrane integrity, mitochondrial membrane potential and normal acrosomes. Anim. Reprod. Sci. 126:23-31.
    Pubmed CrossRef
  3. Anzar M, Boswall L. 2019. Egg yolk-free cryopreservation of bull semen. PLoS One 14:e0223977.
    Pubmed KoreaMed CrossRef
  4. Berry DP, Ring SC, Evans RD. 2020. Choice of artificial insemination beef bulls used to mate with female dairy cattle. J. Dairy Sci. 103:1701-1710.
    Pubmed CrossRef
  5. Birck A, Christensen P, Labouriau R, Borchersen S. 2010. In vitro induction of the acrosome reaction in bull sperm and the relationship to field fertility using low-dose inseminations. Theriogenology 73:1180-1191.
    Pubmed CrossRef
  6. Bousseau S, Brillard JP, Marguant-Le Guienne B, Guérin B, Lechat M. 1998. Comparison of bacteriological qualities of various egg yolk sources and the in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or lecithin based diluents. Theriogenology 50:699-706.
    CrossRef
  7. Butler ST. 2014. Nutritional management to optimize fertility of dairy cows in pasture-based systems. Animal 8 Suppl 1:15-26.
    Pubmed CrossRef
  8. Celeghini EC, de Arruda RP, de Andrade AF, Raphael CF. 2007. Practical techniques for bovine sperm simultaneous fluorimetric assessment of plasma, acrosomal and mitochondrial membranes. Reprod. Domest. Anim. 42:479-488.
    Pubmed CrossRef
  9. Chaveiro A, Machado L, Frijters A, Woelders H. 2006. Improvement of parameters of freezing medium and freezing protocol for bull sperm using two osmotic supports. Theriogenology 65:1875-1890.
    Pubmed CrossRef
  10. Correa JR, Zavos PM. 1997. Relationships among frozen-thawed sperm characteristics assessed via the routine semen analysis, sperm functional tests and fertility of bulls in an artificial insemination program. Theriogenology 48:721-731.
    CrossRef
  11. Correa JR and Zavos PM. 1994. The hypoosmotic swelling test: its employment as an assay to evaluate the functional integrity of the frozen-thawed bovine sperm membrane. Theriogenology 42:351-360.
    CrossRef
  12. Diskin MG. Review: semen handling, time of insemination and insemination technique in cattle. Animal 2018;12(s1):s75-s84.
    Pubmed CrossRef
  13. Farrell PB, Presicce GA, Foote RH. 1998. Quantification of bull sperm characteristics measured by computer-assisted sperm analysis (CASA) and the relationship to fertility. Theriogenology 49:871-879.
    CrossRef
  14. Harrison RA and Vickers SE. 1990. Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. J. Reprod. Fertil. 88:343-352.
    Pubmed CrossRef
  15. Hu CH, Zhuang XJ, Wei YM, Zhang M, Lu SS, Lu YQ, Lu KH. 2017. Comparison of mitochondrial function in boar and bull spermatozoa throughout cryopreservation based on JC-1 staining. Cryo Letters 38:75-79.
    Pubmed
  16. Januskauskas A, Rodriguez-Martinez H. 2003. Subtle membrane changes in cryopreserved bull semen in relation with sperm viability, chromatin structure, and field fertility. Theriogenology 60:743-758.
    Pubmed CrossRef
  17. Jepson A, Arlt J, Statham J, Spilman M, Burton K, Wood T, Martinez VA. 2019. High-throughput characterisation of bull semen motility using differential dynamic microscopy. PLoS One 14:e0202720.
    Pubmed KoreaMed CrossRef
  18. Johnson MS, Senger PL, Allen CH, Hancock DD, Sasser RG. .
  19. Kang SS, Cho SR, Kim UH, Park CS, Kim HC, Chung KY, Lee SD, Jang SS, Jeon G, Kim S, Yang BC. 2016. Analysis of epididymal sperm from Korean native bull (Hanwoo) aged at 8 and 15 months before freezing and after thawing. J. Emb. Trans. 31:109-116.
    CrossRef
  20. Kang SS, Lee MS, Kim UH, Lee SD, Yang BC, Cho SR. 2019. Effect of Optixcell and Triladyl extenders on frozen-thawed sperm motilities and calving rates following artificial insemination in Hanwoo. Korean. J. Agric. Sci. 46:195-204.
  21. Kasai T, Ogawa K, Mizuno K, Nagai S, Uchida Y, Ohta S, Fujie M, Suzuki K, Hoshi K. 2002. Relationship between sperm mitochondrial membrane potential, sperm motility, and fertility potential. Asian J. Androl. 4:97-103.
  22. Kathiravan P, Kalatharan J, Veerapandian C. 2008. Computer automated motion analysis of crossbred bull spermatozoa and its relationship with in vitro fertility in zona-free hamster oocytes. Anim. Reprod. Sci. 104:9-17.
    Pubmed CrossRef
  23. Layek SS, Mohanty TK, Parks JE. 2016. Cryopreservation of bull semen: evolution from egg yolk based to soybean based extenders. Anim. Reprod. Sci. 172:1-9.
    Pubmed CrossRef
  24. Lima FS, Risco CA, Thatcher MJ, Benzaquen ME, Archbald LF, Thatcher WW. 2009. Comparison of reproductive performance in lactating dairy cows bred by natural service or timed artificial insemination. J. Dairy Sci. 92:5456-5466.
    Pubmed CrossRef
  25. López-Gatius F. 2012. Factors of a noninfectious nature affecting fertility after artificial insemination in lactating dairy cows. A review. Theriogenology 77:1029-1041.
    Pubmed CrossRef
  26. Meyers S, Foutouhi A. 2019. Sperm mitochondrial regulation in motility and fertility in horses. Reprod. Domest. Anim. 54 Suppl 3:22-28.
    Pubmed CrossRef
  27. Mocé E, Vicente JS. 2010. Effect of cooling rate to 5 °C, straw size and farm on fertilizing ability of cryopreserved rabbit sperm. Reprod. Domest. Anim. 45:e1-e7.
    Pubmed CrossRef
  28. Morrell JM, Nongbua T, Valeanu S, Lima Verde I, Lundstedt-Enkel K, Johannisson A. 2017. Sperm quality variables as indicators of bull fertility may be breed dependent. Anim. Reprod. Sci. 185:42-52.
    Pubmed CrossRef
  29. Morris GJ. 2006. Rapidly cooled human sperm: no evidence of intracellular ice formation. Hum. Reprod. 21:2075-2083.
    Pubmed CrossRef
  30. Nagata MB, Egashira J, Katafuchi N, Endo K, Ogata K, Yamanaka K, Yamanouchi T, Matsuda H, Yamashita K. 2019. Bovine sperm selection procedure prior to cryopreservation for improvement of post-thawed semen quality and fertility. J. Anim. Sci. Biotechnol. 10:91.
    Pubmed KoreaMed CrossRef
  31. Pace MM and Graham EF. 1974. Components in egg yolk which protect bovine spermatozoa during freezing. J. Anim. Sci. 39:1144-1149.
    Pubmed CrossRef
  32. Senger PL, Mitchell JR, Almquist JO. 1983. Influence of cooling rates and extenders upon post-thaw viability of bovine spermatozoa packaged in .25- and .5-ml French straws. J. Anim. Sci. 56:1261-1268.
    Pubmed CrossRef
  33. Stevenson JS, Jung Y. 2009. Pregnancy outcome after insemination of frozen-thawed bovine semen packaged in two straw sizes: a meta-analysis. J. Dairy Sci. 92:4432-4438.
    Pubmed CrossRef
  34. Valergakis GE, Banos G. 2007. Comparison of artificial insemination and natural service cost effectiveness in dairy cattle. Animal 1:293-300.
    Pubmed CrossRef
  35. Vishwanath R and Shannon P. 2000. Storage of bovine semen in liquid and frozen state. Anim. Reprod. Sci. 62:23-53.
    Pubmed CrossRef
  36. Yang BC, Kang SS, Park CS, Kim UH, Kim HC, Jeon GJ, Kim S, Lee SD, Cho SR. 2015. Motility, fertilizability and subsequent embryonic development of frozen-thawed spermatozoa derived from epididymis in Hanwoo. J. Emb. Trans. 30:271-276.
    CrossRef
  37. Yang DH, Xu ZZ. 2018. Application of liquid semen technology under the seasonal dairy production system in New Zealand. Anim. Reprod. Sci. 194:2-10.
    Pubmed CrossRef

Article

Original Article

Journal of Animal Reproduction and Biotechnology 2020; 35(4): 307-314

Published online December 31, 2020 https://doi.org/10.12750/JARB.35.4.307

Copyright © The Korean Society of Animal Reproduction and Biotechnology.

Spermatozoa motility, viability, acrosome integrity, mitochondrial membrane potential and plasma membrane integrity in 0.25 mL and 0.5 mL straw after frozen-thawing in Hanwoo bull

Sung-Sik Kang , Ui-Hyung Kim , Myung-Suk Lee , Seok-Dong Lee and Sang-Rae Cho*

Hanwoo Research Institute, National Institute Animal Science (NIAS), Rural Development Administration (RDA), Pyeongchang 25340, Korea

Correspondence to:Sang-Rae Cho
E-mail: chosr@korea.kr
ORCID https://orcid.org/0000-0003-0209-6248

Received: October 20, 2020; Revised: November 17, 2020; Accepted: November 30, 2020

Abstract

In the present study, we examined the effect of straw size on spermatozoa motility, viability, acrosome integrity, mitochondrial membrane potential, and plasma membrane integrity after freezing-thawing. Hanwoo semen was collected from three bulls and diluted with an animal protein-free extender, divided into two groups, namely, 10 million spermatozoa in 0.25 mL and 20 million spermatozoa in 0.5 mL straw, and cryopreserved. In Experiment 1, the motility and motility parameters of the frozen-thawed spermatozoa were evaluated. After freezing-thawing, the spermatozoa motility parameters fast progressive, straight line velocity, and average path velocity were compared between the 0.25 mL straw and 0.5 mL straw groups. They were 35.2 ± 1.0 and 32.3 ± 0.7%, 34.6 ± 0.7 and 31.8 ± 0.5 µm/s, 51.4 ± 1.3 and 47.1 ± 1.1 µm/s, 0.25 mL straw and 0.5 mL straw groups, respectively. In Experiment 2, the viability, acrosome membrane integrity, and mitochondrial membrane potential of the frozen-thawed spermatozoa were assessed. After freezing-thawing, the percentages of spermatozoa with live, intact acrosomes and high mitochondrial membrane potential were compared between the in 0.25 mL straw and 0.5 mL straw groups. They were 48.0 ± 2.6% and 35.6 ± 2.8% between the 0.25 mL straw and 0.5 mL straw groups. In Experiment 3, the plasma membrane integrity of frozen-thawed spermatozoa was compared. After freezing-thawing, the plasma membrane integrity was higher for the in 0.25 mL straw group than the 0.5 mL straw group. They were 62.0 ± 2.2 and 54.1 ± 1.3% between the 0.25 mL straw and 0.5 mL straw groups. In conclusion, our results suggest that freezing semen in 0.25 mL straw improves the relative motility, viability, and acrosomal, mitochondrial membrane potential, and plasma membrane integrity of Hanwoo bull spermatozoa.

Keywords: bull, freezing-thawing, spermatozoa, straw size

INTRODUCTION

Natural mating with superior bulls and artificial insemination (AI) using semen collected from elite bulls have been globally applied in cattle breeding (Lima et al., 2009). However, natural mating may spread diseases borne by bulls. The adoption of AI in cattle reproduction mitigates the risk of disease transmission via bulls and lowers breeding costs (Valergakis et al., 2007). Elite bull semen may be transported to other states within the same country and to other countries (López-Gatius, 2012). The desired phenotypes and genes may be transferred to the offspring (Berry et al., 2020). In AI, semen is collected from elite bulls, diluted with suitable semen extenders, and stored in liquid or frozen solid form (Vishwanath and Shannon, 2000). Liquid semen is used in dairy cow reproduction with short (2-mo) short breeding seasons. In Ireland and New Zealand, it is adjusted according to pasture growth (Butler, 2014) and grass nutrient levels (Yang et al., 2018). However, liquid semen in extender can only provide conception rates comparable to those of frozen-thawed semen within the first 3 d (Vishwanath and Shannon, 2000). In contrast, frozen semen is semi-permanently cryopreserved in liquid nitrogen (LN2) and is generally used more often than liquid semen in AI (Layek et al., 2016). In general, semen collected from elite sires is diluted with semen extenders, filled in 0.25 mL or 0.5 mL straw, and cryopreserved in LN2 before use (Diskin, 2018). About 10-30 million spermatozoa are filled in 0.25 mL or 0.5 mL straw, distributed to farms, and used for AI worldwide (Stevenson et al., 2009). The 0.25 mL straw has half the volume of the 0.5 mL straw and more than two 0.25 mL straws can be preserved in the LN2 tank (Johnson et al., 1995). AI technicians and dairy and beef farm owners are often concerned that the low spermatozoa may reduce the conception rate. Nevertheless, there was no reduction in the conception rate after the introduction of 10-30 million spermatozoa for AI (Stevenson et al., 2009).

The Hanwoo Bull Center (Hanwoo Improvement Center, NH, Seosan, Korea) selected elite Hanwoo bulls and produced ~10 million frozen straws per Hanwoo bull lifespan. The Hanwoo Bull Center reported that it packed 18 million spermatozoa per 0.5 mL straw and distributed the frozen straws to all parts of Korea. Asia, Central and South America, and the United States produced 0.5 mL frozen straw. Canada and Europe commercially produced 0.25 mL frozen straw (Diskin, 2018). If 0.25 mL straw is substituted for 0.5 mL straw in semen freezing, the space in the LN2 cryopreservation tank can be doubled. Moreover, frozen straw production time and extender consumption can be reduced. However, the 0.25 mL straw has a larger exposure space than the 0.5 mL straw. Furthermore, the former is more sensitive than the latter to the external LN2 tank temperature and air currents. Therefore, short application time and minimum temperature change are required when 0.25 mL frozen straw is used in AI (Johnson et al., 1995). Despite these precautions, 0.25 mL straw has several advantages over 0.5 mL straw especially when the semen is diluted with tris-citric acid extender supplemented with 20% egg yolk (Anzar et al., 2011). For dairy bulls, frozen-thawed spermatozoa in 0.25 mL straw showed high motility and viability, low incidence of damaged acrosomal membranes (Senger et al., 1983), and high mitochondrial membrane potential compared to frozen-thawed spermatozoa in 0.5 mL straw (Anzar et al., 2011). Until recently, Hanwoo bull semen was only packed in 0.5 mL straw and to the best of our knowledge there were no reports comparing the properties of frozen-thawed spermatozoa in 0.25 mL and 0.5 mL straw.

In the present study, then, we investigated the effects of packing semen in 0.25 mL and 0.5 mL straw after freezing-thawing and compared their relative preservation efficacy. In Experiment 1, we compared the motility and motility parameters of frozen-thawed spermatozoa packed in 0.25 mL and 0.5 mL straw. To this end, we used a computer sperm analysis system. In Experiment 2, we examined the viability, acrosomal membrane integrity, and mitochondrial membrane potential of spermatozoa in 0.25 mL and 0.5 mL straw. For this purpose, we used fluorescent staining after freezing-thawing. In Experiment 3, we applied the hypoosmotic swelling test to study the plasma membrane integrity of spermatozoa in 0.25 mL and 0.5 mL straw after freezing-thawing.

MATERIALS AND METHODS

Bull semen collection and cryopreservation

Bull semen was collected by an electro-ejaculator machine (Electro Jac 6; Neogen Corp., Lansing, MI, USA) between February and March 2020 at the Hanwoo Research Institute, NIAS, RDA, Pyeongchang, Korea. Three bulls aged 15 mo with mean weight 461.0 ± 47.8 kg were used for semen collection. Bulls A and B had two ejaculates each and bull C had one ejaculate. Semen volume, spermatozoa concentration and pH of raw semen were examined before dilution. Spermatozoa motility and motility parameters of raw semen were evaluated after dilution with semen extender and presented in Table 1. Spermatozoa motility was evaluated with a computer-assisted analytical system (CASA; sperm class analyzer; Microoptic SL, Barcelona, Spain). Over 90% of the motile spermatozoa sample was introduced for semen freezing. In brief, the semen was diluted with animal protein-free diluent (Optixcell; IMV Technologies, L’Aigle, France). The spermatozoa concentration was adjusted to 40 × 106 cells/mL and the dilution was cooled in a refrigerator at 4℃ for 3-4 h. The diluted semen was then introduced into 0.25 mL straw (10 × 106 spermatozoa/straw) and 0.5 mL straw (20 × 106 spermatozoa/straw). Spermatozoa density was identical for both the 0.25 mL and 0.5 mL straw. Straws were preserved by immersion for 14 min at 3 cm above the surface of LN2 in a styrofoam box. The straws were then dropped in LN2. The frozen straws were cryopreserved in LN2 tanks until spermatozoa motility and characteristics were evaluated.

Table 1. The basic characteristics of raw semen, spermatozoa motility and motility parameters after dilution.

Raw semen informationMean value (5 replicates)
Volume (mL)3.8 ± 0.7
Sperm concentration (×106 cells/mL)828.5 ± 252.6
pH7.9 ± 0.2
Total motile (%)99.7 ± 0.1
Fast progressive (%)27.0 ± 3.7
Slow progressive (%)65.7 ± 4.3
Non-progressive (%)7.1 ± 1.2
Immotile (%)0.3 ± 0.1
VCL (μm/s)140.3 ± 1.1
VSL (μm/s)39.7 ± 3.2
VAP (μm/s)79.5 ± 5.5
LIN (%)28.6 ± 2.1
STR (%)50.0 ± 2.6
ALH (μm/s)4.0 ± 0.5
BCF (Hz)15.3 ± 0.7

Mean ± SE. Volume, sperm concentration and pH of raw semen were examined after semen collection. Spermatozoa motility and motility parameters were examined after dilution with semen extender. Values indicate mean of 5 replicates from each ejaculates..



Spermatozoa motility and motility parameters after freezing-thawing

Spermatozoa motility was examined as previously described (Yang et al., 2015; Kang et al., 2016), with slight modifications. In brief, frozen semen was immersed in water at 37.5℃ for 40 s and mixed in a 1.5 mL tube. Then 3 μL frozen-thawed semen was introduced to the chamber of a microscope slide (SC 20-01-04-B; Leja, Nieuw-Vennep, Netherlands). At least 800 spermatozoa in 4-5 fields per slide chamber were counted and spermatozoa motility and motility parameters were evaluated by CASA. Spermatozoa motility and the percentages of total motile, fast progressive, slow progressive, non-progressive, and immotile spermatozoa were evaluated. The other spermatozoa motility parameters assessed included curvilinear velocity (VCL, μm/s), straight line velocity (VSL, μm/s), average path velocity (VAP, μm/s), linearity (LIN = VSL/VCL, %), straightness (STR = VSL/VAP, %), amplitude of lateral head (ALH, μm/s), and flagellar beat cross frequency (BCF, Hz).

Spermatozoa viability, acrosomal membrane integrity, and mitochondrial membrane potential of after freezing-thawing

Spermatozoa characteristics were measured according to a previous report (Celeghini et al., 2007), with a modification. Frozen-thawed semen in 0.25 mL and 0.5 mL straw was transferred to a 1.5 mL tube and vortexed for 3 s. One hundred microliters frozen-thawed semen was diluted with 900 μL pre-warmed DPBS (-) at 37℃. One hundred microliters diluted semen was mixed with 150 μL of 5,5’,6,6’-tetrachloro-1,1’,3,3’tetraethylbenzimidazolylcarbocyanine iodide (JC-1; mitochondrial membrane potential detection kit; Cell Technology Inc., Danvers, MA, USA) working solution and incubated for 30 min at 37℃ in the dark. After incubation, 1 μL Hoechst 33342 (H1339; Molecular Probes, Eugene, OR, USA) stock solution was mixed with diluted semen and incubated for 10 min at 37℃ in the dark. Then 1 μL propidium iodide (PI; P-4172; Sigma-Aldrich Crop., St. Louis, MO, USA) stock solution and 1 μL fluorescein peanut agglutinin FITC conjugate (FITC-PNA; FL-1071; Vector Laboratories, Piedmont, Italy) were mixed with diluted semen and incubated for 8 min at 37℃ in the dark. Two microliters of 10% (v/v) formaldehyde was added to the stained semen mixture to impede spermatozoa movement (Harrison and Vickers, 1990). Five microliters stained semen was mounted on a slide glass and covered with a cover slip. More than 200 spermatozoa per microscope slide were counted at × 400 magnification under a fluorescent microscope (Eclipse Ti; Nikon, Tokyo, Japan). Live and dead spermatozoa, acrosomal membrane integrity, and mitochondrial membrane potential were assessed with a triple band filter (DAPI/FITC/TRITC; Nikon, Tokyo, Japan). The heads of live spermatozoa were stained with Hoechst 33342 (blue) while those of dead spermatozoa were stained with PI (red). High mitochondrial membrane potential was indicated by orange JC-1 staining of the spermatozoa midpiece. Low mitochondrial membrane potential was indicated by faint orange or no JC-1 staining of the spermatozoa midpiece. Damaged acrosomal membranes were stained with FITC-PNA (green) at the anterior spermatozoa head while intact acrosomal membranes were not stained at the anterior spermatozoa head.

Fluorescence preparation

One milligram Hoechst 33342 was diluted with 960 μL DPBS (-) and 40 μL dimethyl sulfoxide (DMSO) and cryopreserved at -20℃. The JC-1 stock solution contained 1 mg/mL DMSO in a mitochondrial membrane potential detection kit. To prepare the JC-1 working solution, 5 μL JC-1 stock solution at -20℃ and 500 μL DPBS (-) were mixed and cryopreserved at -20℃. PI stock solution was prepared by mixing 25 mg PI and 1 mL DMSO, diluting this mixture to 2 mg/mL with DPBS (-), and cryopreserving the dilution at -20℃. All fluorescent probe stock solutions except FITC-PNA were stored at -20℃ in the dark. The latter was preserved at 4℃. A 10% (v/v) formaldehyde working solution was prepared by mixing 2.9 mL of 35% (v/v) formaldehyde (Daejung Chemical & Metals Co., Gyeonggi-do, Korea) with 7.1 mL DPBS (-) and storing the dilution at 4℃.

Spermatozoa plasma membrane integrity after freezing-thawing

Spermatozoa plasma membrane integrity was determined as previously described (Kang et al., 2019). After freezing-thawing semen in 0.25 mL and 0.5 mL straw, the thawed semen was transferred to a 1.5 mL tube. Thirty microliters frozen-thawed semen was diluted with 300 μL hypoosmotic swelling test solution (Correa and Zavos, 1994) and incubated for 40 min at 37℃. Five microliters incubated semen was mounted on a glass microscope slide and covered with a cover slip. More than 200 spermatozoa per microscope slide were examined at × 400 magnification and scored as swelling or non-swelling. Swelling spermatozoa were considered to have intact plasma membranes.

Statistical analysis

Motility, motility parameters, viability, acrosomal membrane integrity, mitochondrial membrane potential, and plasma membrane integrity were analyzed by one-way ANOVA followed by Duncan’s test as a post hoc analysis. All analyses were performed in SAS v. 9.2 (SAS Institute, Cary, NC, USA). Spermatozoa viability, acrosomal membrane integrity, and mitochondrial membrane potential were calculated as %. Spermatozoa stained blue on head and orange on midpiece considered to have live, intact acrosomes and high mitochondrial membrane potential (LIAH). Spermatozoa stained blue on head and orange or colorless on midpiece considered to have live, intact acrosomes and low mitochondrial membrane potential (LIAL). Spermatozoa stained red on head considered to have dead, intact acrosomes and low mitochondrial membrane potential (DIAL). Spermatozoa stained red on postal head, green on arterial head, and colorless on midpiece considered to have dead, damaged acrosomes and low mitochondrial membrane potential (DDAL).

RESULTS

Experiment 1

To examine the effects of 0.25 and 0.5 mL straw, we compared frozen-thawed spermatozoa motility in 0.25 mL and 0.5 mL straw. Table 2 shows that the % of fast-progressive spermatozoa in the 0.25 mL straw group was significantly higher than that in the 0.5 mL straw group (35.2 ± 1.0 vs. 32.3 ± 0.7%, respectively; p < 0.05). The % of slow progressive in the 0.25 mL straw group was significantly lower than that in the 0.5 mL straw group (Table 2) (22.9 ± 0.7 vs. 26.0 ± 0.6%. respectively; p < 0.01). The % of VSL in the 0.25 mL straw group was significantly higher than that in 0.5 mL straw group (34.6 ± 0.7 vs. 31.8 ± 0.5%, respectively; p < 0.01). The % of VAP in the 0.25 mL straw group was significantly higher than that in 0.5 mL straw group (51.4 ± 1.3 vs. 47.1 ± 1.1%, respectively; p < 0.05).

Table 2. Spermatozoa motility and motility parameters after freezing-thawing.

Straw size (replicates)

0.25 mL (25)0.5 mL (25)
Total motile (%)92.1 ± 1.388.7 ± 1.3
Fast progressive (%)35.2 ± 1.0a32.3 ± 0.7b
Slow progressive (%)34.0 ± 1.630.4 ± 1.7
Non-progressive (%)22.9 ± 0.7d26.0 ± 0.6c
Immotile (%)7.9 ± 1.311.3 ± 1.3
VCL (μm/s)85.5 ± 2.879.8 ± 2.4
VSL (μm/s)34.6 ± 0.7c31.8 ± 0.5d
VAP (μm/s)51.4 ± 1.3a47.1 ± 1.1b
LIN (%)41.0 ± 0.840.4 ± 0.8
STR (%)67.6 ± 0.868.0 ± 0.9
ALH (μm/s)3.1 ± 0.13.1 ± 0.1
BCF (Hz)15.3 ± 0.314.6 ± 0.1

a,bValues (mean ± SE) with different superscripts in the same row are significantly different (p < 0.05). c,dValues (mean ± SE) with different superscripts in the same row are significantly different (p < 0.01). Five straws of frozen thawed semen were used to evaluate sperm motility per ejaculation group. Values indicate mean of 25 replicates (5 replicates × 5 ejaculates)..



Experiment 2

Spermatozoa viability, acrosomal membrane integrity, and mitochondrial membrane potential after freezing-thawing were examined by the quadruple staining method. Table 3 shows that the % of LIAH in the 0.25 mL straw group was significantly higher than that in the 0.5 mL straw group (48.0 ± 2.6% vs. 35.6 ± 2.8%, respectively; p < 0.01). The % of LIAL in the 0.25 mL straw group was significantly lower than that in 0.5 mL straw group (8.4 ± 0.7 vs. 16.6 ± 1.8%, respectively; p < 0.001).

Table 3. Viability, acrosomal membrane integrity, and mitochondrial membrane potential of spermatozoa after freezing-thawing.

Straw size (mL)ReplicatesLIAHLIALDIALDDAL
0.252548.0 ± 2.6c8.4 ± 0.7f23.1 ± 2.018.6 ± 1.6
0.52535.6 ± 2.8d16.6 ± 1.8e25.7 ± 2.120.4 ± 1.2

c,dValues (mean ± SE) with different superscripts in the same column are significantly different (p < 0.01). e,fValues (mean ± SE) with different superscripts in the same column are significantly different (p < 0.001). Values indicate mean of 25 replicates (5 replicates × 5 ejaculates). Spermatozoa with live intact acrosome and high mitochondrial membrane integrity, LIAH; spermatozoa with live intact acrosome and low mitochondrial membrane integrity, LIAL; spermatozoa with dead intact acrosome and low mitochondrial membrane integrity, DIAL; spermatozoa with dead damaged acrosome and low mitochondrial membrane integrity, DDAL; five straws of frozen-thawed semen were used to evaluate spermatozoa characteristics per ejaculation group..



Experiment 3

The differences in plasma membrane integrity between the 0.25 mL and 0.5 mL straw groups are shown in Table 4. The % of intact plasma membranes in the 0.25 mL straw group was significantly higher than that in the 0.5 mL straw (62.0 ± 2.2 vs. 54.1 ± 1.3%, respectively, p < 0.01).

Table 4. Plasma membrane integrity of spermatozoa after freezing-thawing.

Straw size (mL)ReplicatesIntact plasma membrane (%)*
0.252562.0 ± 2.2c
0.52554.1 ± 1.3d

c,dValues (mean ± SE) with different superscripts in the same column are significantly different (p < 0.01). Asterisk indicates % intact plasma membrane spermatozoa. Five straws of frozen-thawed semen were used to evaluate intact plasma membrane spermatozoa per ejaculation group. Swollen spermatozoa were scored as spermatozoa with intact plasma membranes..


DISCUSSION

The sizes of the straws used to freeze-thaw bull semen vary with continent and country. Canada and Europe have adopted 0.25 mL straw while the United States and South America have adopted 0.5 mL straw. The optimal straw size and fertility rate are disputed among herdsman and technicians (Stevenson et al., 2009). In Korea, bull semen has been cryopreserved in 0.5 mL straw with semen extender based on tris-citric acid supplemented with 20% egg yolk. The latter has been used as a cryoprotectant in semen extender (Pace and Graham, 1974). However, animal proteins derived from egg yolk has disadvantages such as bacterial and mycoplasmal contamination (Bousseau et al., 1998). Recently, animal protein-free semen extenders have been investigated (Anzar et al., 2019). Here, we used an animal protein-free extender for Hanwoo bull semen. To the best of our knowledge, this work is the first to compare Hanwoo spermatozoa characteristics after freezing-thawing in 0.25 mL and 0.5 mL straw containing animal protein-free extender. We determined the relative effects of two different straw sizes on spermatozoa motility, viability, acrosomal membrane integrity, mitochondrial membrane potential, and plasma membrane integrity after freezing-thawing.

Spermatozoa motility and motility parameters have been used to predict fertility in livestock animals (Jepson et al., 2019). The percentages of fast progressive, VSL, and VAP for spermatozoa after thawing in 0.25 mL straw were significantly higher than those for spermatozoa after thawing in 0.5 mL straw (Table 2). These findings are consistent with a previous report showing that the fast progressive, VSL, and VAP of spermatozoa in 0.25 mL straw were higher than those of spermatozoa in 0.5 mL straw after 2 h thawing (Anzar et al., 2011). Enhanced fast progressive sperm motility in frozen-thawed bull semen increased its relative fertility (Morrell et al., 2017). The VSL and VAP were highly correlated with fertility in vitro (Kathiravan et al., 2008) and with fertility prediction in vivo (Farrell and Brockett, 1998). These earlier studies demonstrated that spermatozoa quality in 0.25 mL straw after freezing-thawing was superior to that of spermatozoa after freezing-thawing in 0.5 mL straw. Semen freezing in 0.25 mL straw augments post-AI fertility compared to that for semen freezing in 0.5 mL straw.

The proportion of spermatozoa with live, intact acrosome membranes, and high mitochondrial membrane potential (LIAH) was ~12% higher in 0.25 mL straw than it was in 0.5 mL straw (Table 3). Previous reports indicated higher LIAH for spermatozoa in 0.25 mL straw than those in 0.5 mL straw. An elevated proportion of LIAH may improve relative fertility (Anzar et al., 2011). Ansari et al. (2011) reported that the 0.25 mL straw group had 14% more viable spermatozoa than the 0.5 mL straw. The acrosome reaction is essential for fertilization. Cryopreservation and thawing induce the acrosome reaction in spermatozoa (Birck et al., 2010). The reduction of the acrosome reaction in spermatozoa during freezing and thawing is critical for increasing the relative proportion of spermatozoa with intact acrosomes (Nagata et al., 2019). Mitochondrial membrane potential is a predictor of in spermatozoa fertility potential in boars and bulls (Hu et al., 2017), stallions (Meyers et al., 2019), and humans (Kasai et al., 2002). The plasma membrane integrity of spermatozoa in 0.25 mL straw was 7.9% higher that of spermatozoa in 0.5 mL straw after freezing-thawing (Table 4). Elevated plasma membrane integrity of spermatozoa is an important indicator of bull fertility (Correa et al., 1997; Januskauskas et al., 2003). Hence, we propose that freezing bull semen in 0.25 mL straw increases spermatozoa motility, viability, acrosome integrity, mitochondrial membrane potential, and plasma membrane integrity.

In the present study, we assessed spermatozoa motility by CASA and spermatozoa characteristics by fluorescent staining and HOST. Reductions in spermatozoa quality by semen dilution, freezing, and thawing processes are inevitable in frozen semen fabrication (Chaveiro, 2006). As it has a large surface-to-volume, the 0.25 mL straw induces faster cooling and thawing than the 0.5 mL straw (Mocé et al., 2010). Cooling acceleration in 0.25 mL straw causes spermatozoa to pass through the fracture temperature and minimizes intracellular crystallization (Morris, 2006). However, the relatively improved motility, viability, acrosome membrane integrity, mitochondrial membrane potential, and plasma membrane integrity of the spermatozoa in 0.25 mL straw in vitro does not necessarily guarantee enhanced conception rates in vivo after AI. In fact, AI with 0.25 mL straw conferred only a 0.74% improvement in the expected fertility relative to AI using 0.5 mL straw (Stevenson et al., 2009). The use of 0.25 mL and 0.5 mL straw varies with country and continent and fertility rates vary with AI technician skills and straw handling (Diskin, 2018). The 0.25 mL straw is sensitive to exposure time, thawing conditions, and transportation after freezing-thawing (Stevenson et al., 2009). Future studies should evaluate the post-AI fertility rates using Hanwoo frozen-thawed spermatozoa in 0.25 mL straw. Moreover, the effects of freezing and thawing in 0.25 mL straw on spermatozoa characteristics should also be examined.

CONCLUSION

The present study demonstrated that semen freezing in 0.25 mL straw improved relative spermatozoa motility, viability, acrosome integrity, mitochondrial membrane potential, and plasma membrane integrity after freezing-thawing. The use of 0.25 mL straw doubles the number of straws that can be processed simultaneously in the LN2 tank compared to the 0.5 mL straws. Nevertheless, the in vivo post-AI fertility rates obtained with 0.25 mL straw should be investigated.

ACKNOWLEDGEMENTS

This study received support from the Cooperative Research Program for Agriculture Science & Technology Development project: “Development of improved technologies for preservation of Hanwoo bull semen and technology application”, PJ0143252020, RDA, Korea, and the 2020 Postdoctoral Fellowship Program of the Hanwoo Research Institute, NIAS, RDA, Korea.

We thank Mr. In-Rak Yoon, Mr. Woo-Heon Choi, Mr. Jong-Hwan Jang, Mr. Kyu-Myung Kim who care for bulls, supporting of semen collection and semen cryopreservation in Hanwoo Research Institute.

CONFLICTS OF INTEREST


No potential conflict of interest relevant to this article was reported.

AUTHOR CONTRIBUTIONS


Conceptualization: Kang SS, Cho SR

Data curation: Kang SS

Formal analysis: Kang SS

Funding acquisition: Cho SR, Kim UH

Investigation: Kang SS, Lee MS, Lee SD

Methodology: Kang SS, Lee MS, Lee SD

Project administration: Cho SR

Resources: Cho SR

Software: Kang SS

Supervision: Cho SR

Validation: Kang SS, Cho SR, Kim UH

Visualization: Kang SS

Writing - original draft: Kang SS

Writing - review & editing: Kang SS

AUTHOR’S POSITION AND ORCID NO.


SS Kang, Postdoctoral Fellow, https://orcid.org/0000-0002-9453-5377

UH Kim, Researcher, https://orcid.org/0000-0002-2197-5080

MS Lee, Research Assistant, https://orcid.org/0000-0002-9111-8535

SD Lee, Research Assistant, https://orcid.org/0000-0002-4481-5338

SR Cho, Researcher, https://orcid.org/0000-0003-0209-6248

Table 1 . The basic characteristics of raw semen, spermatozoa motility and motility parameters after dilution.

Raw semen informationMean value (5 replicates)
Volume (mL)3.8 ± 0.7
Sperm concentration (×106 cells/mL)828.5 ± 252.6
pH7.9 ± 0.2
Total motile (%)99.7 ± 0.1
Fast progressive (%)27.0 ± 3.7
Slow progressive (%)65.7 ± 4.3
Non-progressive (%)7.1 ± 1.2
Immotile (%)0.3 ± 0.1
VCL (μm/s)140.3 ± 1.1
VSL (μm/s)39.7 ± 3.2
VAP (μm/s)79.5 ± 5.5
LIN (%)28.6 ± 2.1
STR (%)50.0 ± 2.6
ALH (μm/s)4.0 ± 0.5
BCF (Hz)15.3 ± 0.7

Mean ± SE. Volume, sperm concentration and pH of raw semen were examined after semen collection. Spermatozoa motility and motility parameters were examined after dilution with semen extender. Values indicate mean of 5 replicates from each ejaculates..


Table 2 . Spermatozoa motility and motility parameters after freezing-thawing.

Straw size (replicates)

0.25 mL (25)0.5 mL (25)
Total motile (%)92.1 ± 1.388.7 ± 1.3
Fast progressive (%)35.2 ± 1.0a32.3 ± 0.7b
Slow progressive (%)34.0 ± 1.630.4 ± 1.7
Non-progressive (%)22.9 ± 0.7d26.0 ± 0.6c
Immotile (%)7.9 ± 1.311.3 ± 1.3
VCL (μm/s)85.5 ± 2.879.8 ± 2.4
VSL (μm/s)34.6 ± 0.7c31.8 ± 0.5d
VAP (μm/s)51.4 ± 1.3a47.1 ± 1.1b
LIN (%)41.0 ± 0.840.4 ± 0.8
STR (%)67.6 ± 0.868.0 ± 0.9
ALH (μm/s)3.1 ± 0.13.1 ± 0.1
BCF (Hz)15.3 ± 0.314.6 ± 0.1

a,bValues (mean ± SE) with different superscripts in the same row are significantly different (p < 0.05). c,dValues (mean ± SE) with different superscripts in the same row are significantly different (p < 0.01). Five straws of frozen thawed semen were used to evaluate sperm motility per ejaculation group. Values indicate mean of 25 replicates (5 replicates × 5 ejaculates)..


Table 3 . Viability, acrosomal membrane integrity, and mitochondrial membrane potential of spermatozoa after freezing-thawing.

Straw size (mL)ReplicatesLIAHLIALDIALDDAL
0.252548.0 ± 2.6c8.4 ± 0.7f23.1 ± 2.018.6 ± 1.6
0.52535.6 ± 2.8d16.6 ± 1.8e25.7 ± 2.120.4 ± 1.2

c,dValues (mean ± SE) with different superscripts in the same column are significantly different (p < 0.01). e,fValues (mean ± SE) with different superscripts in the same column are significantly different (p < 0.001). Values indicate mean of 25 replicates (5 replicates × 5 ejaculates). Spermatozoa with live intact acrosome and high mitochondrial membrane integrity, LIAH; spermatozoa with live intact acrosome and low mitochondrial membrane integrity, LIAL; spermatozoa with dead intact acrosome and low mitochondrial membrane integrity, DIAL; spermatozoa with dead damaged acrosome and low mitochondrial membrane integrity, DDAL; five straws of frozen-thawed semen were used to evaluate spermatozoa characteristics per ejaculation group..


Table 4 . Plasma membrane integrity of spermatozoa after freezing-thawing.

Straw size (mL)ReplicatesIntact plasma membrane (%)*
0.252562.0 ± 2.2c
0.52554.1 ± 1.3d

c,dValues (mean ± SE) with different superscripts in the same column are significantly different (p < 0.01). Asterisk indicates % intact plasma membrane spermatozoa. Five straws of frozen-thawed semen were used to evaluate intact plasma membrane spermatozoa per ejaculation group. Swollen spermatozoa were scored as spermatozoa with intact plasma membranes..


References

  1. Ansari MS, Rakha BA, Akhter S. 2011. Effect of straw size and thawing time on quality of cryopreserved buffalo (Bubalus bubalis) semen. Reprod. Biol. 11:49-54.
    Pubmed CrossRef
  2. Anzar M, Boswall L. 2011. Cryopreservation of bull semen shipped overnight and its effect on post-thaw sperm motility, plasma membrane integrity, mitochondrial membrane potential and normal acrosomes. Anim. Reprod. Sci. 126:23-31.
    Pubmed CrossRef
  3. Anzar M, Boswall L. 2019. Egg yolk-free cryopreservation of bull semen. PLoS One 14:e0223977.
    Pubmed KoreaMed CrossRef
  4. Berry DP, Ring SC, Evans RD. 2020. Choice of artificial insemination beef bulls used to mate with female dairy cattle. J. Dairy Sci. 103:1701-1710.
    Pubmed CrossRef
  5. Birck A, Christensen P, Labouriau R, Borchersen S. 2010. In vitro induction of the acrosome reaction in bull sperm and the relationship to field fertility using low-dose inseminations. Theriogenology 73:1180-1191.
    Pubmed CrossRef
  6. Bousseau S, Brillard JP, Marguant-Le Guienne B, Guérin B, Lechat M. 1998. Comparison of bacteriological qualities of various egg yolk sources and the in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or lecithin based diluents. Theriogenology 50:699-706.
    CrossRef
  7. Butler ST. 2014. Nutritional management to optimize fertility of dairy cows in pasture-based systems. Animal 8 Suppl 1:15-26.
    Pubmed CrossRef
  8. Celeghini EC, de Arruda RP, de Andrade AF, Raphael CF. 2007. Practical techniques for bovine sperm simultaneous fluorimetric assessment of plasma, acrosomal and mitochondrial membranes. Reprod. Domest. Anim. 42:479-488.
    Pubmed CrossRef
  9. Chaveiro A, Machado L, Frijters A, Woelders H. 2006. Improvement of parameters of freezing medium and freezing protocol for bull sperm using two osmotic supports. Theriogenology 65:1875-1890.
    Pubmed CrossRef
  10. Correa JR, Zavos PM. 1997. Relationships among frozen-thawed sperm characteristics assessed via the routine semen analysis, sperm functional tests and fertility of bulls in an artificial insemination program. Theriogenology 48:721-731.
    CrossRef
  11. Correa JR and Zavos PM. 1994. The hypoosmotic swelling test: its employment as an assay to evaluate the functional integrity of the frozen-thawed bovine sperm membrane. Theriogenology 42:351-360.
    CrossRef
  12. Diskin MG. Review: semen handling, time of insemination and insemination technique in cattle. Animal 2018;12(s1):s75-s84.
    Pubmed CrossRef
  13. Farrell PB, Presicce GA, Foote RH. 1998. Quantification of bull sperm characteristics measured by computer-assisted sperm analysis (CASA) and the relationship to fertility. Theriogenology 49:871-879.
    CrossRef
  14. Harrison RA and Vickers SE. 1990. Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. J. Reprod. Fertil. 88:343-352.
    Pubmed CrossRef
  15. Hu CH, Zhuang XJ, Wei YM, Zhang M, Lu SS, Lu YQ, Lu KH. 2017. Comparison of mitochondrial function in boar and bull spermatozoa throughout cryopreservation based on JC-1 staining. Cryo Letters 38:75-79.
    Pubmed
  16. Januskauskas A, Rodriguez-Martinez H. 2003. Subtle membrane changes in cryopreserved bull semen in relation with sperm viability, chromatin structure, and field fertility. Theriogenology 60:743-758.
    Pubmed CrossRef
  17. Jepson A, Arlt J, Statham J, Spilman M, Burton K, Wood T, Martinez VA. 2019. High-throughput characterisation of bull semen motility using differential dynamic microscopy. PLoS One 14:e0202720.
    Pubmed KoreaMed CrossRef
  18. Johnson MS, Senger PL, Allen CH, Hancock DD, Sasser RG. .
  19. Kang SS, Cho SR, Kim UH, Park CS, Kim HC, Chung KY, Lee SD, Jang SS, Jeon G, Kim S, Yang BC. 2016. Analysis of epididymal sperm from Korean native bull (Hanwoo) aged at 8 and 15 months before freezing and after thawing. J. Emb. Trans. 31:109-116.
    CrossRef
  20. Kang SS, Lee MS, Kim UH, Lee SD, Yang BC, Cho SR. 2019. Effect of Optixcell and Triladyl extenders on frozen-thawed sperm motilities and calving rates following artificial insemination in Hanwoo. Korean. J. Agric. Sci. 46:195-204.
  21. Kasai T, Ogawa K, Mizuno K, Nagai S, Uchida Y, Ohta S, Fujie M, Suzuki K, Hoshi K. 2002. Relationship between sperm mitochondrial membrane potential, sperm motility, and fertility potential. Asian J. Androl. 4:97-103.
  22. Kathiravan P, Kalatharan J, Veerapandian C. 2008. Computer automated motion analysis of crossbred bull spermatozoa and its relationship with in vitro fertility in zona-free hamster oocytes. Anim. Reprod. Sci. 104:9-17.
    Pubmed CrossRef
  23. Layek SS, Mohanty TK, Parks JE. 2016. Cryopreservation of bull semen: evolution from egg yolk based to soybean based extenders. Anim. Reprod. Sci. 172:1-9.
    Pubmed CrossRef
  24. Lima FS, Risco CA, Thatcher MJ, Benzaquen ME, Archbald LF, Thatcher WW. 2009. Comparison of reproductive performance in lactating dairy cows bred by natural service or timed artificial insemination. J. Dairy Sci. 92:5456-5466.
    Pubmed CrossRef
  25. López-Gatius F. 2012. Factors of a noninfectious nature affecting fertility after artificial insemination in lactating dairy cows. A review. Theriogenology 77:1029-1041.
    Pubmed CrossRef
  26. Meyers S, Foutouhi A. 2019. Sperm mitochondrial regulation in motility and fertility in horses. Reprod. Domest. Anim. 54 Suppl 3:22-28.
    Pubmed CrossRef
  27. Mocé E, Vicente JS. 2010. Effect of cooling rate to 5 °C, straw size and farm on fertilizing ability of cryopreserved rabbit sperm. Reprod. Domest. Anim. 45:e1-e7.
    Pubmed CrossRef
  28. Morrell JM, Nongbua T, Valeanu S, Lima Verde I, Lundstedt-Enkel K, Johannisson A. 2017. Sperm quality variables as indicators of bull fertility may be breed dependent. Anim. Reprod. Sci. 185:42-52.
    Pubmed CrossRef
  29. Morris GJ. 2006. Rapidly cooled human sperm: no evidence of intracellular ice formation. Hum. Reprod. 21:2075-2083.
    Pubmed CrossRef
  30. Nagata MB, Egashira J, Katafuchi N, Endo K, Ogata K, Yamanaka K, Yamanouchi T, Matsuda H, Yamashita K. 2019. Bovine sperm selection procedure prior to cryopreservation for improvement of post-thawed semen quality and fertility. J. Anim. Sci. Biotechnol. 10:91.
    Pubmed KoreaMed CrossRef
  31. Pace MM and Graham EF. 1974. Components in egg yolk which protect bovine spermatozoa during freezing. J. Anim. Sci. 39:1144-1149.
    Pubmed CrossRef
  32. Senger PL, Mitchell JR, Almquist JO. 1983. Influence of cooling rates and extenders upon post-thaw viability of bovine spermatozoa packaged in .25- and .5-ml French straws. J. Anim. Sci. 56:1261-1268.
    Pubmed CrossRef
  33. Stevenson JS, Jung Y. 2009. Pregnancy outcome after insemination of frozen-thawed bovine semen packaged in two straw sizes: a meta-analysis. J. Dairy Sci. 92:4432-4438.
    Pubmed CrossRef
  34. Valergakis GE, Banos G. 2007. Comparison of artificial insemination and natural service cost effectiveness in dairy cattle. Animal 1:293-300.
    Pubmed CrossRef
  35. Vishwanath R and Shannon P. 2000. Storage of bovine semen in liquid and frozen state. Anim. Reprod. Sci. 62:23-53.
    Pubmed CrossRef
  36. Yang BC, Kang SS, Park CS, Kim UH, Kim HC, Jeon GJ, Kim S, Lee SD, Cho SR. 2015. Motility, fertilizability and subsequent embryonic development of frozen-thawed spermatozoa derived from epididymis in Hanwoo. J. Emb. Trans. 30:271-276.
    CrossRef
  37. Yang DH, Xu ZZ. 2018. Application of liquid semen technology under the seasonal dairy production system in New Zealand. Anim. Reprod. Sci. 194:2-10.
    Pubmed CrossRef