Journal Search Engine
Search Advanced Search Adode Reader(link)
Download PDF Export Citaion korean bibliography PMC previewer
ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.55 No.6 pp.65-73
DOI : https://doi.org/10.14397/jals.2021.55.6.65

Effect of Lecithin Hanwoo Semen after Freeze-Thawing

Sung-Sik Kang, Ui-Hyung Kim, Kyung-Woon Kim, Sang-Rae Cho*†
Hanwoo Research Institute, National Institute Animal Science, Rural Development Administration, Pyeongchang 25340, Korea

These authors contributed equally to this work.


* Corresponding author: Sang-Rae Cho Tel: +82-33-330-0625 Fax: +82-33-330-0660 E-mail: chosr@korea.kr
October 28, 2021 November 17, 2021 December 8, 2021

Abstract


In the present study, we examined the effect of different lecithin concentrations on spermatozoa characteristics after freeze-thawing. Hanwoo semen was collected from one bull and divided into five groups (tris-citric acid semen extender with 0, 0.1, 0.25, and 0.5% lecithin groups as well as a 20% egg yolk group). Semen extender with 20% egg yolk was used as control treatment. After the freeze-thawing of semen, spermatozoa motility, motility parameters, viability, acrosomal membrane integrity, mitochondrial membrane potential, and plasma membrane integrity were examined. In experiment 1, the effect of different lecithin concentrations on spermatozoa motility and associated parameters was examined. The 0.1% lecithin-treated spermatozoa showed greater fast progressive motility (%) in addition to higher VCL (μm/s), VSL (μm/s), and VAP (μm/s) when compared to other lecithin concentration groups and controls. In experiment 2, the effect of different lecithin concentrations on spermatozoa viability was examined. The 0.1% and 0.25% lecithin addition groups (55.4±7.3 and 51.7±11.2%) exhibited similar viability compared to the control group (54.1±12.6%). In experiment 3, the effects of different lecithin concentrations on viability, acrosomal membrane integrity, and mitochondrial membrane integrity of spermatozoa were examined. The percentage of live spermatozoa with an intact acrosome and high mitochondrial membrane potential in the 0.1% lecithin group was not significantly different compared to the control group (31.2±13.3 vs. 30.5±10.9%). In experiment 4, the effect of different lecithin concentrations on the plasma membrane integrity of spermatozoa was examined. The percentage of spermatozoa with a normal plasma membrane was similar between the 0.1% lecithin and control groups (31.2±13.3 vs. 30.5±10.9%). In conclusion, we suggest that semen extender supplemented with 0.1% lecithin can replace 20% egg yolk without reducing spermatozoa quality.



초록


    Introduction

    Artificial insemination (AI) of cattle has been employed in the dairy and beef industries for the generation of genetically elite offspring using selected elite bulls (Moore & Hasler, 2017). The AI method has several merits for farm management, as the expected delivery date, precise bull number, and expected heritability are known in advance (Vishwanath, 2003). In general, the fresh semen of selected elite bulls is collected via artificial vaginas and electroejaculation (Barth et al., 2004). To produce semen suitable for AI, collected semen is diluted with extenders and preserved in a liquid state or frozen in liquid nitrogen (LN2) (Vishwanath & Shannon, 2000). Frozen semen is used in almost all countries that perform AI for reproduction (Diskin, 2018). Liquid semen has also been applied for cattle reproduction in some countries, such as Ireland (Murphy et al., 2017) and New Zealand (Yang et al., 2018), which have a short breeding season of two–three months. To preserve semen in the frozen state, it is generally diluted with appropriate semen extenders supplemented with egg yolk (Moore and Hasler, 2017). However, egg yolk supplementation of semen extenders carries a risk of bacterial and mycoplasma infection (Bousseau et al., 1998). Therefore, alternative plant-derived supplements are being explored. In particular, lecithin is a phosphatidylcholine-containing phospholipid mixture reported as an alternative to animal-derived egg yolk (Layek et al., 2016). Thus, numerous studies have evaluated different lecithin concentrations in semen extender. In goats, the addition of 2% lecithin to semen extender improved spermatozoa motility and plasma membrane integrity after preservation at 5℃ for 2 days (Silva et al., 2019). In rams, 1% lecithin- supplemented semen extender improved the motility and viability of frozen-thawed spermatozoa (Forouzanfar et al., 2010) as also observed for 1.5% lecithin (Emamverdi et al., 2013) compared to 20% egg yolk supplementation. 1.5% lecithin was considered an optimal concentration for frozen- thawed spermatozoa motility when compared to 20% egg yolk-supplemented semen extender in goats (Salmani et al., 2014) and rabbits (Nishijima et al., 2015). In buffalo, the addition of 10% lecithin-supplemented semen extender improved fertility after AI with frozen-thawed semen compared to egg yolk supplementation (Akhter et al., 2012). In bulls, 1.5% lecithin supplementation resulted in the highest spermatozoa quality after preservation in tris-based extender (Tarig et al., 2017). Motility and viability are critical indicators of spermatozoa quality in bulls (Januskauskas et al., 1996). Aires et al. (2003) reported that semen frozen with a commercial soy lecithin extender increased frozen-thawed spermatozoa motility and Holstein bull pregnancy rate compared to a commercial tris egg yolk extender. In buffalo, the addition of 10% lecithin to semen extender improved frozen-thawed spermatozoa motility and fertility compared to semen extender supplemented with 20% egg yolk (Akhter et al., 2012).

    In contrast, lecithin supplementation in semen extender was reported to reduce the mitochondrial membrane potential of spermatozoa after freeze-thawing in rams (Del Valle et al., 2012). While spermatozoa mitochondrial membrane potential is correlated to spermatozoa motility and fertility, the optimal lecithin concentration in semen extender after freeze-thawing still has not been evaluated in bulls (Kasai et al., 2002;Silva et al., 2019). In canines, semen extender with 1% lecithin improved mitochondrial membrane and plasma membrane integrity after chilling. Of note, plasma membrane integrity is also used as an indicator of spermatozoa quality (Bassiri et al., 2012). Overall, the appropriate lecithin concentration in semen extenders differed between studies.

    Further, the effect of lecithin supplementation on Hanwoo bull semen still has not been evaluated. Therefore, we aimed to assess the effect of different lecithin concentrations in tris-citric acid-based semen extender on spermatozoa motility, viability, acrosomal membrane integrity, mitochondrial membrane potential, and plasma membrane integrity after freezethawing Hanwoo bull semen.

    Materials and Methods

    For this study, we purchased commercially available lecithin (L-a-phosphatidylcholine, Sigma, p3644).

    1. Semen collection, freezing, and thawing

    Semen was collected from one Hanwoo bull (age: 21 months; weight: 518 kg) using an artificial vagina at the Hanwoo Research Institute of the National Institute of Animal Science at Pyeongchang. After semen collection, ejaculates were transferred to the laboratory within one min. Ejaculate volume, pH, and motility were then examined. Semen with a minimum of 80% motility was used for the experiments. The semen volume was 2.0 to 4.5 ml, and the pH was 6.0 to 7.5. Semen was divided into five groups as follows: 0, 0.1, 0.25, and 0.5% of lecithin, and 20% egg yolk in tris-critic acid extender. The composition of semen extenders is described in Table 1. Sperm concentration was adjusted to 15 to 20 million spermatozoa/0.5 ml straw. Sperm was cooled in a refrigerator at 4℃ for 4 h. Cooled semen was introduced into a 0.5 ml straw in a cold chamber at 4℃, put 3 cm above the liquid nitrogen (LN2) surface for 14 min, and cryopreserved in an LN2 tank. After 6 months, frozen straws were thawed in water at 37℃ for 40 s and used for the experiments.

    2. Examination of spermatozoa motility and associated parameters after freeze-thawing

    Spermatozoa motility was examined as previously described (Kang et al., 2020). Frozen-thawed semen was mixed in a 1.5 ml tube, and 2 μl semen was introduced into a count chamber (4 chamber slide-20 μm, ref SC 20-01-04-B, Leja, Netherlands) on a slide warmer at 37℃. After one min, spermatozoa motility and motility parameters were evaluated using a computer-assisted sperm analyzer (sperm class analyzer, Microptic, Spain). The total motile (%), fast progressive (%), slow progressive (%), low progressive (%), non-motile (%), and hyperactive (%) spermatozoa as well as the curvilinear velocity (VCL, μm/s), straight line velocity (VSL, μm/s), average path velocity (VAP, μm/s), linearity of a curvilinear path (LIN, %), linearity of the average path (STR, %), amplitude of lateral head displacement (ALH, μm), and beat cross frequency (BCF, Hz) were evaluated.

    3. Examination of spermatozoa viability after freeze-thawing

    The viability of spermatozoa was evaluated using a cell counter (Nucleocounter SP-100, Chemometec, Denmark). Briefly, frozen-thawed semen was diluted with lysis buffer (Reagent S100, Chemometec, Denmark). All spermatozoa were stained with propidium iodide (PI), and the total number of spermatozoa was examined. To evaluate non-viable spermatozoa, frozen-thawed semen was diluted with PBS (ⅹ20). Only dead spermatozoa were stained with PI, and the number of non-viable spermatozoa was evaluated. Viability was calculated according to the following formula: Viability (%) = [(total number of spermatozoa – number of non-viable spermatozoa) / (total number of spermatozoa)] × 100

    4. Examination of viability, acrosomal membrane integrity, and mitochondrial membrane potential of frozen-thawed spermatozoa

    Spermatozoa motility was examined as previously described (Kang et al., 2020). Briefly, frozen-thawed semen was diluted 10× with DPBS (Sigma, D4031). Thereafter, 50 μl of diluted semen and 150 μl of JC-1 working solution were mixed and incubated at 37℃ for 30 min in the dark. Two μl of Hoechst 33342 was added to the semen mixture and incubated at 37℃ for 10 min in the dark. One μl of PI and FITC-PNA was added to the mixture and incubated at 37℃ for 8 min in the dark. Seven μl of stained semen was mounted on a glass slide and covered with a coverslip. 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 using 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 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.

    5. Evaluation of spermatozoa plasma membrane integrity after freeze-thawing

    Spermatozoa plasma membrane integrity was determined as previously described (Kang et al., 2020). After freeze-thawing semen in 0.25 ml and 0.5 ml straws, the thawed semen was transferred to a 1.5 ml tube. Thirty microliters freeze-thawed semen was diluted with 300 μl hypoosmotic swelling test solution (Correa & Zavos, 1994) and incubated for 1 h at 37℃. Seven μl of incubated semen was mounted on a glass microscope slide and covered with a coverslip. More than 200 spermatozoa per microscope slide were examined at × 400 magnification and identified as swelling or non-swelling. Swelling spermatozoa were considered to have intact plasma membranes.

    6. Statistical analysis

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

    Results and Discussion

    As shown in Table 2, the percentage of total motile spermatozoa in the 0.1% lecithin group was significantly higher than those in the 0 and 0.5% lecithin groups (73.8±15.4 vs. 61.3±17.8 and 57.2±25.3%, p<0.001), but similar to that in the control group. The percentage of fast progressive spermatozoa in the 0.1% lecithin group (18.6±6.6%) was significantly higher than in the 0, 0.25, and 0.5% lecithin groups as well as the control group (14.7±6.4, 11.7±5.9, 7.2±5.0, and 14.7±5.3%, respectively, p<0.001).

    The percentages of VCL, VSL, and VAP in the 0.1% lecithin group were significantly improved compared to those in the remaining lecithin groups and the control group (p<0.001). The percentage of LIN in the control group (34.1±3.8%) was significantly higher than that in the 0.1, 0.25, and 0.5% lecithin groups (31.1±3.6, 28.2±5.2, and 26.3±5.8%, respectively, p<0.001). The percentage of STR in the 0.25 and 0.5% lecithin groups (53.1±8.5% and 51.2±8.5%) was significantly lower than in the 0 and 0.1% lecithin groups as well as the control group (58.5±5.8, 56.8±6.7, and 59.2±5.1%, respectively, p<0.001). The percentage of ALH in the 0.1% lecithin group (3.7±0.3 μm) was higher than in the 0.25 and 0.5% lecithin group as well as the control group (3.3±0.6, 2.8±0.9, and 2.7±0.4 μm, respectively, p<0.001). The percentage of BCF in the 0.1% lecithin group (14.5±1.0 Hz) was significantly higher compared to those in the 0, 0.25, and 0.5% lecithin groups, and the control group (13.2±1.4, 11.7±1.7, 9.9±2.8, and 12.2±1.6 Hz, respectively, p<0.001).

    The viability of frozen-thawed spermatozoa at different lecithin concentrations is shown in Table 3. The percentage of viable spermatozoa in the 0.1% lecithin group was significantly higher than in the 0 and 0.5% lecithin groups (39.1±9.8% and 46.5±12.0%) as well as the control group (54.1±12.6%, p<0.001).

    As shown in Table 4, the percentage of LIAH in the 0.1% lecithin group (31.2±13.3%) was significantly higher than that in 0 and 0.5% lecithin groups (19.0±9.5% and 17.2±13.0%), while similar to that in the control group (30.5±10.9%, p<0.001). The percentage of LIAL in the 0.5% lecithin group was significantly higher than in the 0, 0.1, and 0.25% lecithin groups and control group (p<0.001).

    As shown in Table 5, the percentages of normal plasma membranes in the 0.1% lecithin and control group were similar (31.2±13.3% and 30.5±10.9%, p<0.001). However, the lecithin 0 and 0.5% groups showed a significant decrease in the percentage of normal plasma membranes of frozen-thawed spermatozoa (19.0±9.5% and 17.2±13.0%) compared to the 0.1% lecithin group and the control group (31.2±13.3% and 30.5±10.9%, p<0.0001)

    Soybean lecithin is an alternative to egg yolk from animal sources as a semen extender supplement, eliminating the risk of bacterial and mycoplasma contamination. In the present study, we evaluated different lecithin concentrations in semen extender to confirm the optimal appropriate lecithin concentration for Hanwoo bull AI. We evaluated the motility, plasma membrane integrity, viability, acrosomal membrane integrity, and mitochondrial membrane potential of Hanwoo bull spermatozoa after freeze-thawing. In our preliminary study, 1, 3, 5, and 10% lecithin supplementation in semen extender exhibited deleterious effects on spermatozoa motility and viability compared to 20% egg yolk supplementation in tris-citric acid extender (data not shown).

    Thus, we examined 0.1, 0.25, and 0.5% lecithin supplementation in semen extender for bull semen freezing to confirm the appropriate lecithin alternative to 20% egg yolk. The addition of 0.1 to 0.25% lecithin in semen extender both improved the percentage of total motility, resulting in similar percentages of total motility (Table 2). 0.1% lecithin supplementation in semen extender resulted in the highest percentage of fast progressive spermatozoa after freeze-thawing among all groups evaluated. A higher percentage of total motility and fast progressive motility is associated with the improvement of pregnancy rate (Allouche et al., 2017) as well as the in vitro fertilization rate in bulls (Li et al., 2016).

    Evaluation of spermatozoa motility parameters revealed that the addition of 0.1% lecithin to semen extender increased the VCL, VSL, and VAP of frozen-thawed spermatozoa compared to other lecithin concentration groups and the control group. After freeze-thawing semen, higher VCL, VSL, and VAP are considered indicators of improved fertility in bulls based on the CASA evaluation system (Gillan et al., 2008).

    The percentage of LIN in the 0.1% lecithin group was lower than that in the other lecithin groups and the control group. A lower LIN of spermatozoa is associated with reduced fertility (Freour et al., 2012). However, the reduction of LIN in the 0.1% lecithin group may falsely suggest decreased fertility, as the respective percentages of VCL and VSL were significantly higher than those in the other lecithin groups and the control group. Thus, we considered the decreased LIN in the 0.1% lecithin group to be a numerical value error resulting from the division of a low percentage of VSL by VCL. Further studies are needed to explore the association of fertility with a low percentage of LIN, resulting from such numerical value expression errors.

    In the present study, 0.1% lecithin supplementation improved both the ALH and BCF compared to the other lecithin concentrations and control treatment. Higher ALH and BCF were reported as strongly correlated with sperm hyperactivation and fertility in bulls (Farrell et al., 1998) In turn, an increase in hyperactivated spermatozoa allows for easier oocyte penetration (Chamberland et al., 2001).

    Therefore, we suggest that 0.1% lecithin in tris-citric acid-based extender is the optimal concentration for improving frozen-thawed spermatozoa motility and motility parameters, highlighting the utility of 0.1% lecithin as an alternative to 20% egg yolk supplementation during Hanwoo bull in vitro fertilization. Akhter et al. (2012) assessed the effect of different lecithin concentrations (5, 10, and 15%) in semen extender on buffalo spermatozoa, reporting that 10% lecithin improved spermatozoa viability after freeze-thawing to a greater extent compared to 20% egg yolk.

    Thus, they suggested that the addition of 10% lecithin in semen extender is an alternative to 20% egg yolk supplementation and observed improved fertility after AI. Herein, we assumed that the differences in optimal lecithin concentrations between the Hanwoo bull and buffalo arise from differences in the sensitivity of spermatozoa between cattle species. To verify the effect of different lecithin concentrations on spermatozoa motility, lecithin of different sources and semen samples from different bull species were assessed. Thus, the optimal lecithin concentrations in semen extender varied. It has been reported that the addition of 1% lecithin in tris-based semen extender is optimal for frozen-thawed spermatozoa quality in goats (Chelucci et al., 2015), humans (Reed et al., 2009), rams (Forouzanfar et al., 2010), and roosters (Mehdipour et al., 2018).

    In the present study, we confirmed that 1% lecithin and 0.1% lecithin supplementation in semen extender were effective in improving frozen-thawed spermatozoa motility. Further, the addition of 0.1% lecithin in semen extender resulted in similar spermatozoa viability compared to the control treatment. The plasma membrane integrity of frozen-thawed spermatozoa was similar between 0.1 and 0.25% supplementation.

    The viability of frozen-thawed spermatozoa was previously described as positively correlated with motility (Januskauskas et al., 2000), and greater plasma membrane integrity was positively correlated with fertility after AI in bulls (Maxwell et al., 1996). Lecithin exerts its beneficial effects by protecting the outer plasma membrane of cells from damage induced by cryopreservation and thawing processes (Fiume, 2001), playing an important role in cell membrane reorganization (Sych et al., 2018).

    Thus, we concluded that 0.1% lecithin supplementation in tris-citric acid-based semen extender is sufficient to protect the plasma membrane and subsequently increase the viability of spermatozoa after freeze-thawing. In addition, 0.1% lecithin supplementation increased the percentage of live spermatozoa with high mitochondrial membrane potential and intact acrosomes. There has been no report on the appropriate lecithin concentration to improve Hanwoo bull spermatozoa after freeze-thawing. of note, a previous study revealed that supplementation with lecithin reduced the mitochondrial membrane potential of spermatozoa from rams after freeze-thawing (Del Valle et al., 2012).

    However, the group only reported the effect of 3.5% lecithin in semen extender, as other concentrations were not examined. In the present study, we also confirmed that 0.5% lecithin increased the number of live spermatozoa with intact acrosomes and low mitochondrial membrane potential. Further, 0.1% lecithin in semen extender improved the percentage of live spermatozoa with intact acrosomes and high mitochondrial membrane potential. The high mitochondrial membrane potential of spermatozoa has been considered an indicator of viability and motility (Silva & Gadella, 2006). In addition, spermatozoa with high mitochondrial membrane potential have high binding affinity for the oviduct, thus enhancing fertility (Saraf et al., 2017).

    Intact acrosomes are essential for normal fertilization processes, such as capacitation, the acrosome reaction, and fertility (Thomas et al., 1997;Saraf et al., 2019). Taken together, we suggest that 0.1% lecithin is a viable alternative to 20% egg yolk for semen extender supplementation without reducing the mitochondrial membrane potential or compromising the acrosomal membrane integrity of spermatozoa after freeze-thawing.

    In conclusion, In the present study, we determined the optimal lecithin concentration in tris-citric acid-based semen extender without reduction in spermatozoa quality relative to 20% egg yolk supplementation. The addition of 0.1% lecithin in semen extender improved the motility, viability, plasma membrane integrity, acrosomal membrane integrity, and mitochondrial membrane integrity of Hanwoo bull spermatozoa to a greater extent compared to 20% egg yolk. Further studies should evaluate the relationship between freeze-thawed spermatozoa preserved with 0.1% lecithin and the pregnancy rate after AI in this bull species.

    Acknowledgement

    This study was conducted with the support of the Cooperative Research Program for Agriculture Science & Technology Development project: “Development of improved technologies for preservation of Hanwoo bull semen and technology application”, PJ0143252021, RDA, Korea, and the 2021 Postdoctoral Fellowship Program of the Hanwoo Research Institute, NIAS, RDA, Korea. We thank In-Rak Yoon, Woo-Heon Choi, Jong-Hwan Jang, and Kyu-Myung Kim at the Hanwoo Research Institute, NIAS, RDA, Korea for their assistance with bulls’ semen collection and freezing.

    Figure

    Table

    Composition of semen extenders

    Effect of different lecithin concentrations on Hanwoo bull spermatozoa motility after freeze-thawing

    Effect of different lecithin concentrations on spermatozoa viability after freeze-thawing

    Effect of different lecithin concentrations on viability, acrosomal membrane integrity, and mitochondrial membrane potential of spermatozoa after freeze-thawing

    Effect of different lecithin concentrations on plasma membrane integrity of spermatozoa after freeze-thawing

    Reference

    1. Aires VA , Hinsch KD , Mueller-Schloesser F , Bogner K , Mueller-Schloesser S and Hinsch E. 2003. In vitro and in vivo comparison of egg yolk-based and soybean lecithin- based extenders for cryopreservation of bovine semen. Theriogenology 60: 269-279.
    2. Akhter S, Ansari MS, Andrabi SM, Rakha BA, Ullah N and Khalid M.2012. Soya-lecithin in extender improves the freezability and fertility of buffalo (Bubalus bubalis) bull spermatozoa. Reprod. Domest. Anim. 47: 815-819.
    3. Allouche L , Madani T , Mechmeche M , Clement L and Bouchemal A. 2017. Bull fertility and its relation with density gradient selected sperm. Int. J. Fertil. Steril. 11: 55-62.
    4. Barth AD , Arteaga AA , Brito LF and Palmer CW. 2004. Use of internal artificial vaginas for breeding soundness evaluation in range bulls: an alternative for electroejaculation allowing observation of sex drive and mating ability. Anim. Reprod. Sci. 84: 315-325.
    5. Bassiri F , Tavalaee M , Shiravi AH , Mansouri S and Nasr-Esfahani MH. 2012. Is there an association between HOST grades and sperm quality? Hum. Reprod. 27: 2277-2284.
    6. Bousseau S , Brillard JP , Marguant-Le Guienne B , Guerin B , Camus A and 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.
    7. Chamberland A , Fournier V , Tardif S , Sirard MA , Sullivan R and Bailey JL. 2001. The effect of heparin on motility parameters and protein phosphorylation during bovine sperm capacitation. Theriogenology 55: 823-835.
    8. Chelucci S , Pasciu V , Succu S , Addis D , Leoni GG , Manca ME , Naitana S and Berlinguer F. 2015. Soybean lecithin- based extender preserves spermatozoa membrane integrity and fertilizing potential during goat semen cryopreservation. Theriogenology 83: 1064-1074.
    9. 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.
    10. Del Valle I , Gomez-Duran A , Holt WV , Muino-Blanco T and Cebrian-Perez JA. 2012. Soy lecithin interferes with mitochondrial function in frozen-thawed ram spermatozoa. J. Androl. 33: 717-725.
    11. Diskin MG. 2018. Review: Semen handling, time of insemination and insemination technique in cattle. Anim. 12(s1): s75-s84.
    12. Emamverdi M , Zhandi M , Zare Shahneh A , Sharafi M and Akbari-Sharif A. 2013. Optimization of ram semen cryopreservation using a chemically defined soybean lecithin- based extender. Reprod. Domest. Anim. 48: 899-904.
    13. Farrell PB , Presicce GA , Brockett CC and Foote RH. 1998. Quantification of bull sperm characteristics measured by computer-assisted sperm analysis (CASA) and the relationship to fertility. Theriogenology 49: 871-879.
    14. Fiume Z. 2001. Final report on the safety assessment of Lecithin and Hydrogenated Lecithin. Int J. Toxicol. 20 (Suppl 1): 21-45.
    15. Forouzanfar M , Sharafi M , Hosseini SM , Ostadhosseini S , Hajian M , Hosseini L , Abedi P , Nili N , Rahmani HR and Nasr-Esfahani MH. 2010. In vitro comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of ram semen. Theriogenology 73: 480-487.
    16. Freour T , Jean M , Mirallie S and Barriere P. 2012. Computer-assisted sperm analysis parameters in young fertile sperm donors and relationship with age. Syst. Biol. Reprod. Med. 58: 102-106.
    17. Gillan L , Kroetsch T , Maxwell WM and Evans G. 2008. Assessment of in vitro sperm characteristics in relation to fertility in dairy bulls. Anim. Reprod. Sci. 103: 201-214.
    18. Januskauskas A , Haard MG , Haard MC , Soderquist L , Lundeheim N and Rodriguez-Martinez H. 1996. Estimation of sperm viability in frozen-thawed semen from Swedish A.I. bulls. Zentralbl. Veterinarmed. A. 43: 281-287.
    19. Januskauskas A , Johannisson A , Soderquist L and Rodriguez-Martinez H. 2000. Assessment of sperm characteristics post-thaw and response to calcium ionophore in relation to fertility in Swedish dairy AI bulls. Theriogenology 53: 859-875.
    20. Kang SS , Kim UH , Lee MS , Lee SD and Cho SR. 2020. 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. J. Anim. Reprod. Bitotech. 35: 307-314.
    21. Kasai T , Ogawa K , Mizuno K , Nagai S , Uchida Y , Ohta S , Fujie M , Suzuki K , Hirata S and Hoshi K. 2002. Relationship between sperm mitochondrial membrane potential, sperm motility, and fertility potential. Asian J. Androl. 4: 97-103.
    22. Layek SS , Mohanty TK , Kumaresan A and Parks JE. 2016. Cryopreservation of bull semen: Evolution from egg yolk based to soybean based extenders. Anim. Reprod. Sci. 172: 1-9.
    23. Li Y , Kalo D , Zeron Y and Roth Z. 2016. Progressive motility-a potential predictive parameter for semen fertilization capacity in bovines. Zygote 24: 70-82.
    24. Maxwell WM , Welch GR and Johnson LA. 1996. Viability and membrane integrity of spermatozoa after dilution and flow cytometric sorting in the presence or absence of seminal plasma. Reprod. Fertil. Dev. 8: 1165-1178.
    25. Mehdipour M , Daghigh Kia H , Moghaddam G and Hamishehkar H. 2018. Effect of egg yolk plasma and soybean lecithin on rooster frozen-thawed sperm quality and fertility. Theriogenology 116: 89-94.
    26. Moore SG and Hasler JF. 2017. A 100-year review: Reproductive technologies in dairy science. J. Dairy. Sci. 100: 10314-10331.
    27. Murphy EM , Murphy C , O'Meara C , Dunne G , Eivers B , Lonergan P and Fair S. 2017. A comparison of semen diluents on the in vitro and in vivo fertility of liquid bull semen. J. Dairy. Sci. 100: 1541-1554.
    28. Nishijima K , Kitajima S , Koshimoto C , Morimoto M , Watanabe T , Fan J and Matsuda Y. 2015. Motility and fertility of rabbit sperm cryopreserved using soybean lecithin as an alternative to egg yolk. Theriogenology 84: 1172-1175.
    29. Reed ML , Ezeh PC , Hamic A , Thompson DJ and Caperton CL. 2009. Soy lecithin replaces egg yolk for cryopreservation of human sperm without adversely affecting postthaw motility, morphology, sperm DNA integrity, or sperm binding to hyaluronate. Fertil. Steril. 92: 1787-1790.
    30. Salmani H , Towhidi A , Zhandi M , Bahreini M and Sharafi M. 2014. In vitro assessment of soybean lecithin and egg yolk based diluents for cryopreservation of goat semen. Cryobiology 68: 276-280.
    31. Saraf KK , Kumaresan A , Chhillar S , Nayak S , Lathika S , Datta TK , Gahlot SC , Karan P , Verma K and Mohanty TK. 2017. Spermatozoa with high mitochondrial membrane potential and low tyrosine phosphorylation preferentially bind to oviduct explants in the water buffalo (Bubalus bubalis). Anim. Reprod. Sci. 180: 30-36.
    32. Saraf KK , Singh RK , Kumaresan A , Nayak S , Chhillar S , Lathika S , Datta TK and Mohanty TK. 2019. Sperm functional attributes and oviduct explant binding capacity differs between bulls with different fertility ratings in the water buffalo (Bubalus bubalis). Reprod. Fertil. Dev. 31: 395-403.
    33. Silva PF and Gadella BM. 2006. Detection of damage in mammalian sperm cells. Theriogenology 65: 958-978.
    34. Silva R , Batista AM , Arruda LCP , de Souza HM , Nery I , Gomes WA , Soares PC , Silva SV and Guerra MMP. 2019. Concentration of soybean lecithin affects short-term storage success of goat semen related with seminal plasma removal. Anim. Reprod. 16: 895-901.
    35. Sych T , Mely Y and Romer W. 2018. Lipid self-assembly and lectin-induced reorganization of the plasma membrane. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 373: 20170117.
    36. Tarig AA , Wahid H , Rosnina Y , Yimer N , Goh YM , Baiee FH , Khumran AM , Salman H , Assi MA and Ebrahimi M. 2017. Effect of different concentrations of soybean lecithin and virgin coconut oil in Tris-based extender on the quality of chilled and frozen-thawed bull semen. Vet. World. 10: 672-678.
    37. Thomas CA , Garner DL , DeJarnette JM and Marshall CE. 1997. Fluorometric assessments of acrosomal integrity and viability in cryopreserved bovine spermatozoa. Biol. Reprod. 56: 991-998.
    38. Vishwanath R and Shannon P. 2000. Storage of bovine semen in liquid and frozen state. Anim. Reprod. Sci. 62: 23-53.
    39. Vishwanath R. 2003. Artificial insemination: the state of the art. Theriogenology 59: 571-584.
    40. Yang DH , Standley NT and Xu ZZ. 2018. Application of liquid semen technology under the seasonal dairy production system in New Zealand. Anim. Reprod. Sci. 194: 2-10.
    오늘하루 팝업창 안보기 닫기
    오늘하루 팝업창 안보기 닫기