Protocol Optimization for Long‐Term Liquid Storage of Goat Semen in a Chemically Defined Extender

A specific problem in the preservation of goat semen has been the detrimental effect of seminal plasma on the viability of spermatozoa in extenders containing egg yolk or milk. The use of chemically defined extenders will have obvious advantages in liquid storage of buck semen. Our previous study showed that the self‐made mZAP extender performed better than commercial extenders, and maintained a sperm motility of 34% for 9 days and a fertilizing potential for successful pregnancies for 7 days. The aim of this study was to extend the viability and fertilizing potential of liquid‐stored goat spermatozoa by optimizing procedures for semen processing and storage in the mZAP extender. Semen samples collected from five goat bucks of the Lubei White and Boer breeds were diluted with the extender, cooled and stored at 5°C. Stored semen was evaluated for sperm viability parameters, every 48 h of storage. Data from three ejaculates of different bucks were analysed for each treatment. The percentage data were arcsine‐transformed before being analysed with anova and Duncan’s multiple comparison test. While cooling at the rate of 0.1–0.25°C/min did not affect sperm viability parameters, doing so at the rate of 0.6°C/min from 30 to 15°C reduced goat sperm motility and membrane integrity. Sperm motility and membrane integrity were significantly higher in semen coated with the extender containing 20% egg yolk than in non‐coated semen. Sperm motility, membrane integrity and acrosomal intactness were significantly higher when coated semen was 21‐fold diluted than when it was 11‐ or 51‐fold diluted and when extender was renewed at 48‐h intervals than when it was not renewed during storage. When goat semen coated with the egg yolk‐containing extender was 21‐fold diluted, cooled at the rate of 0.07–0.25°C/min, stored at 5°C and the extender renewed every 48 h, a sperm motility of 48% was maintained for 13 days, and an in vitro‐fertilizing potential similar to that of fresh semen was maintained for 11 days.     

Effect of Different Extenders on In Vitro Characteristics of Feline Epididymal Sperm During Cryopreservation

To evaluate and compare the efficacy of various extenders for the cryopreservation of epididymal cat spermatozoa, two experiments were planned. Bovine and equine commercial extenders in the experiment 1 and TRIS–egg yolk–based extenders in experiment 2 were separately studied since the number of sperm collected per cat is reduced. Epididymal sperm samples were packaged into 0.25‐ml straws and frozen. Vigour, motility, morphology, acrosome status, sperm viability and functional membrane integrity were assessed at collection, after cooling and after thawing, while DNA integrity was evaluated at 0‐ and 6‐h post‐thaw. Experiment 1 compared the effect of three non‐feline commercial extenders – based on TRIS–egg yolk (Triladyl), egg‐yolk‐free medium (AndroMed) and skimmed milk‐egg yolk (Gent) – on the quality of frozen‐thawed epididymal cat sperm. Values for sperm motility and functional membrane integrity in cooled sperm diluted in Triladyl were higher (p < 0.001) than those recorded for Andromed and Gent. Except sperm morphology, the other assessed characteristics showed significant higher values in frozen‐thawed sperm diluted in Triladyl than in Andromed and Gent extenders. Experiment 2 analysed the effects of three TRIS–egg yolk–based extenders, one non‐feline commercial (Triladyl) and the other two prepared using different monosaccharides (glucose and fructose), on freezing‐thawed sperm. Results showed that specifically prepared extenders for cryopreservation of feline spermatozoa performed better than the commercial extender Triladyl, although sperm quality during the freezing‐thawing process did not significantly differ associated with the type of monosaccharide (glucose vs fructose) added to the mentioned extenders. Although TRIS–egg yolk–based extenders prepared in experiment 2 improved sperm cryoprotection, Triladyl remains a good option for practitioners who, for ease of use and availability, prefer to work with commercial extenders.     

Influence of the Preservation Temperature (37, 20, 4, −196°C) and the Mixing of Semen over Sperm Quality of Majorera Bucks

This study assessed the effect of different semen storage temperatures and the influence of semen pooling in semen viability. In experiment 1, semen samples (n = 30) of five Majorera bucks were individually processed [Individual semen (IS)] and after the first dilution (Tris‐yolk extender), semen‐diluted aliquots from each male were pooled semen (PS). Thereafter, semen samples (IS and PS) were preserved as fresh semen (37 and 20°C), chilled semen (4°C) and frozen semen. Sperm motility and the percentage of abnormal sperm cells and intact membrane acrosomes were defined. Semen preservation at 20 and 4°C did not modify the quality of spermatozoa for the first 24 h, but the conservation at 37°C caused a dramatic fall in the semen motility from 12 h onwards. Furthermore, the longevity of frozen‐thawed semen was limited to 4–6 h. No differences were observed in semen parameters when PS was compared with semen from individual males in any of the preservation protocols assessed. In experiment 2, 120 goats were distributed in four experimental groups: in group fresh individual semen (FIS, n = 30) and group frozen‐thawed individual semen (FTIS, n = 30), does were transcervically inseminated with fresh semen and frozen‐thawed semen from each individual male, respectively, and in group fresh pooled semen (FPS, n = 30) and group frozen‐thawed pooled semen (FTPS, n = 30), goats were transcervically inseminated with FPS and FTPS, respectively. The kidding rate was very close in the FIS and FPS groups (70.0% and 73.7%, respectively), and no significant differences were observed in the fertility rate between FTIS and FTPS. The results of this study confirmed that semen samples may be preserved satisfactorily for 24 h both at 20 and 4°C. In addition, the mixture of semen of different bucks did not significantly modify the semen parameters when compared with semen from individual males.     

Use of Glutamine and Low Density Lipoproteins Isolated from Egg Yolk to Improve Buck Semen Freezing

To improve the results obtained with a reference cryopreservation extender (control extender: Triladyl® + 20% (v/v) egg yolk + 6.4% (v/v) glycerol) for freezing caprine semen, glutamine was added to 18 split ejaculates at concentrations of 0, 20, 40, 80 and 120 m m (experiment 1). In experiment 2, glutamine was added to 18 split ejaculates at concentrations of 20, 25, 30, 35 and 40 m m . In the third experiment, the egg yolk was replaced with the low-density lipoprotein (LDL) fraction of egg yolk. The quality of frozen then thawed spermatozoa in each extender was compared using computer-assisted semen analysis. In experiment 1, glutamine at concentrations of 20 m m and 40 m m significantly improved sperm motility compared with the control extender. However, at 120 m m , a significant decrease in motility and velocity was observed. In experiment 2, motility, curvilinear velocity and amplitude of lateral head displacement (ALH) were improved in glutamine at 25 m m compared with the control. In experiment 3, 8% LDL and 25 m m glutamine significantly improved sperm motility, straight line velocity and ALH. In the fourth experiment, the quality of the previously defined freezing extender (Triladyl® + 8% (v/v) LDL + 25 m m glutamine + 6.4% (v/v) glycerol) was tested by comparing acrosome, tail membrane, plasma membrane and DNA integrity in 18 split ejaculates of frozen then thawed spermatozoa with spermatozoa that had been frozen then thawed in the control extender, and with spermatozoa from fresh, unfrozen sperm. The percentage of spermatozoa with intact acrosomes and tail membranes was significantly higher with the newly defined extender than that observed with the control extender. There was no significant difference in the percentage of spermatozoa with intact DNA between the frozen and fresh semen.     

Influence of Cool Storage before Freezing on the Quality of Frozen–Thawed Semen Samples in Dogs

The aim of this study was to determinate the semen quality of frozen–thawed samples that were chilled for up to 2 days before freezing. The ejaculates (n = 18) from six dogs were collected, pooled and divided into six aliquots. The first aliquot (C, control) was frozen in liquid nitrogen using a conventional protocol to reach a final concentration of 100 × 10⁶ spermatozoa/ml, 20% egg yolk and 5% glycerol. The remaining five aliquots were diluted with a chilled extender (Tris‐glucose and 20% egg yolk) and cooled at 4°C as follows: R1, the semen was cooled for 1 h; R6, the semen was cooled for 6 h; R12, the semen was cooled for 12 h; R24, the semen was cooled for 24 h and R48, the semen was cooled for 48 h. After the chilling period, a second extender was added (Tris‐glucose, 20% egg yolk, 10% glycerol and Equex at 1%) to reach a final composition similar to aliquot C, and then, the semen samples (R1, R6, R12, R24 and R48) were frozen in liquid nitrogen. The post‐thaw sperm quality was assessed in 30 straws from each experimental group. After freezing–thawing, the total sperm motility (approximately 60–70%) in the semen chilled for up to 48 h did not show any differences from the samples frozen by the conventional cryopreservation method (63.2%). No significant differences were detected in the percentages of abnormal sperm cells among the fresh semen, the control group and the frozen samples after the different cooling times. Finally, the post‐thaw percentages of damaged acrosomes showed a very uniform distribution, with mean values ranging between 7% and 10.5%. The results clearly demonstrated that cooling the semen up to 48 h before freezing did not produce a decrease in the semen quality when was compared with semen frozen by a traditional procedure.     

Fertility Results after Vaginal Deposition of Frozen‐Thawed Buck Semen Diluted with Two Different Extenders Using One‐ or Two‐Step Procedures

In two field trials (T1 and T2), the effect of two different extenders for buck semen was tested. Semen from six (T1) and seven (T2) bucks of the Norwegian Dairy Goat breed was diluted either in a milk‐based extender containing egg yolk (M) or in a commercially available extender without egg yolk [Andromed^® (A)]. Dilution in M was performed in a two‐step procedure including centrifugation of the ejaculates and removal of the supernatant, while dilution in A was performed in one step. During the two trials (T1 and T2) 514 and 714, does, respectively, were artificially inseminated during natural oestrus, and the farmers performed the inseminations themselves after attending an artificial insemination (AI) training course. Vaginal insemination with 200 × 10⁶ spermatozoa diluted in M resulted in a 25‐day non‐return rate (NRR) and kidding rate of 37.3% and 24.5%, respectively, while semen diluted in A resulted in 31.7% NRR and a kidding rate of 19.8% (T1). In T2, NRR and kidding rate for AI performed with semen diluted with M were 42.7% and 28.5%, respectively, while dilution in A gave 37.2% NRR and a kidding rate of 26.8%. There was no significant effect of extender in the two trials [T1:p = 0.068 (NRR), p = 0.148 (kidding rate), T2:p = 0.096 (NRR), p = 0.38 (kidding rate)], but farmer had a significant effect on the fertility parameters in both trials. In conclusion, the present studies may indicate that Andromed^® is suitable for buck semen. However, more research is necessary to confirm the results and to improve the fertility of does after vaginal AI with frozen‐thawed semen.     

Effect of adding different levels of caffeine in the extender on some biochemical constituents,enzymatic activities and physical characteristics of chilled and frozen ram semen

Two experiments were carried out to determine the efficiency of supplementation of ram semen extender with caffeine on chilled storage and frozen capacity of spermatozoa. In the first experiment, eighty ejaculates were collected by an artificial vagina from five adult Barki rams, aged 2–3 years and weighted 45.0 ± 2.0 kg throughout the experimental period (January to February 2017). The ejaculates were pooled and diluted (1:10) with tris‐citric egg yolk extender and were split into five groups. Group 1 served as control, whereas groups 2‐5 were supplemented with 0.1, 0.2, 0.3 and 0.4 mM caffeine. All diluted semen specimens were evaluated for physical characteristics immediately after dilution (T₀) and throughout preservation period of 48 hr at 4°C. Simultaneously, oxidative stress and indices such as total antioxidant capacity (TAC), malondialdehyde concentrations (MDA) and alkaline transaminase (AKP) concentrations and value of resazurin reduction test (RRT) were determined. In the second experiment, the raw pooled ejaculates were diluted (1:10) with glycerolated tris‐citric egg yolk extender, receiving the previously mentioned caffeine levels. The post‐thaw assessment of cryopreserved spermatozoa, in all groups, was conducted by a computer‐assisted sperm analysis (CASA) system. The results revealed that adding caffeine to ram semen extender at low (0.1 mM) or medium (0.2 mM) levels had positive impact on both physical characteristics of ram sperm and the enzymatic activities compared to the other semen groups. Caffeine supplementation also enhanced post‐thaw sperm dynamics, which implies its potential as an exogenous antioxidant supplement.     

Influence of different anaesthetic protocols over the sperm quality on the fresh,chilled (4°C) and frozen‐thawed epididymal sperm samples in domestic dogs

This study assessed the influence of three different anaesthetic protocols on semen quality obtained from the epididymis. Sixty male dogs undergoing to routine sterilization were assigned to three anaesthetic protocols: thiopental group (TG, n = 20), propofol group (PG, n = 20) and ketamine–dexmedetomidine group (KDG, n = 20). Immediately after orchidectomy, the cauda epididymides and vas deferent ducts were isolated and then a retrograde flushing was performed to collect spermatozoa. In experiment 1, after the initial evaluation of the semen (sperm concentration, sperm motility and the percentages of live spermatozoa, abnormal spermatozoa and acrosome membrane integrity), semen samples were diluted in Tris‐glucose‐egg yolk extender and chilled for 48 hr, and the sperm motility was assessed at 6, 24 and 48 hr. In experiment 2, semen samples were diluted in Tris‐glucose‐egg yolk extender and chilled for 24 hr, and then samples were frozen in two extenders with different glycerol concentrations, to reach a final concentration of 50–100 × 10⁶ spermatozoa ml^?1, 20% egg yolk, 0.5% Equex and 4% and 5% glycerol, respectively. Mean values of total sperm concentration, sperm viability and the percentages of intact acrosome and abnormal spermatozoa were not significantly different between experimental groups, and therefore, the anaesthetic protocols assessed did not affect sperm parameters mentioned above. However, our study confirmed a detrimental effect of the use of thiopental (TG) over the total sperm motility (p < 0.05) and progressive sperm motility (p < 0.05) of the fresh and chilled epididymal sperm samples. The anaesthetic protocols including the application of propofol or ketamine–dexmedetomidine can be used to recover sperm in domestic canids without significant changes in sperm quality compared when semen is collected routinely and these techniques could be applicable to endangered wild canids.     

Chilled storage of ram semen improves with the addition of egg yolk and glycerol to milk-based extenders

This study aimed at comparing in vitro, ultra‐heat‐treated (UHT) skim milk and INRA‐96^®‐based extenders supplemented or not with 5% egg yolk and/or 2% glycerol on sperm quality parameters along 72 h of preservation at 5°C, using a factorial design. Semen from six healthy mature Merino rams was pooled and extended in each medium using a split sample procedure (six replicates) and chilled. Subjective motility (SM) (%), membrane integrity (MI) (%) and uncapacitated spermatozoa (US) (×10⁶ spermatozoa/AI dose) were used to assess the semen quality at 0, 12, 24, 48 and 72 h of preservation. UHT‐based extenders yielded better (p < 0.05) SM and MI than INRA‐96^®‐based extenders (59.7% vs 57.9%; 60.2% vs 55.8%, respectively) but similar numbers of US (64.2% vs 62.3 × 10⁶ sperm/AI dose, respectively) along the preservation time. Egg yolk–glycerol or just egg yolk as additives improved (p < 0.05) the results compared with the base extenders without additives or just with glycerol. The sperm parameters assessed decline slowly from 0 to 48 h, with a sharp decline (p < 0.05) at 72 h of preservation. In conclusion, UHT and INRA‐96^® were similar as base extenders, and the addition of 5% egg yolk plus 2% glycerol or just 5% egg yolk improved the quality of ram semen preserved at 5°C, at least for 48 h. The combination of egg yolk–glycerol might provide extra protection in case of fluctuation of temperatures below 5°C, commonly seen under field conditions.     

Cryopreservation of Dog Semen in a Tris Extender with 1% or 2% Soya Bean Lecithin as a Replacement of Egg Yolk

Egg yolk is usually included in extenders used for preservation of dog semen. Lecithin is an interesting animal‐protein free alternative to egg yolk for semen preservation. The aim of our study was to evaluate soya bean lecithin for cryopreservation of dog semen. Five ejaculate replicates were divided in three equal parts, centrifuged and each pellet diluted with one of the three Tris‐based extenders containing 20% egg yolk, 1% soya bean lecithin or 2% soya bean lecithin. Extended semen was loaded in 0.5‐ml straws, cooled and diluted a second time and frozen in liquid nitrogen vapours. Sperm motility parameters (CASA), acrosome integrity (FITC‐PNA/PI) and sperm membrane integrity (C‐FDA) were evaluated 5 min post‐thaw and after 2 and 4 h of incubation. Total motility was significantly better in the egg yolk extender than in any of the lecithin‐based extender and was better in the 1% lecithin extender than in the 2% lecithin extender. Sperm membrane integrity was significantly better in the egg yolk extender than in any of the lecithin‐based extenders but did not differ significantly between the 1% and 2% lecithin extenders. Acrosome integrity was significantly better in the egg yolk extender than in the 2% lecithin extender but did not differ between the egg yolk extender and the 1% lecithin extender or between the two lecithin extenders. In conclusion, egg yolk was superior to lecithin in our study. The extender with 1% lecithin preserved sperm motility better than the extender with 2% lecithin.     

Melatonin can improve viability and functional integrity of cooled and frozen/thawed rabbit spermatozoa

Melatonin is known to protect sperm against freezing-inflicted damage in different domestic species. The aim of the study was to evaluate the effect of supplementation of semen extender with melatonin on the quality and DNA integrity of cooled and frozen/thawed rabbit spermatozoa. We also investigated whether the addition of melatonin to the semen extender could improve the fertility of rabbit does artificially inseminated with frozen/thawed semen. Semen samples collected from eight rabbit bucks were pooled and then diluted in INRA-82 supplemented either with (0.5, 1.0 or 1.5 mM) or without (0.0 mM) melatonin. Diluted semen was cooled at 5°C for 24 hr. For cryopreservation and based on the first experiment's best result, semen samples were diluted in INRA-82 in the presence or absence of 1.0 mM melatonin and then frozen in 0.25 ml straws. Following cooling or thawing, sperm quality and DNA integrity were evaluated. Furthermore, the fertility of frozen/thawed semen was investigated after artificial insemination. Supplementation of semen extender with 1.0 mM melatonin improved (p < .05) motility, viability, membrane and acrosome integrities in cooled semen compared with other groups. Sperm quality and DNA integrity were higher (p < .05) in frozen/thawed semen diluted in 1.0 mM melatonin-supplemented extender than in the control group. Conception and birth rates were higher in does inseminated with 1.0 mM melatonin treated semen compared with the controls. In conclusion, supplementation of semen extender with 1.0 mM melatonin improved the quality of cooled and frozen/thawed rabbit spermatozoa. Melatonin can preserve DNA integrity and enhance the fertility of frozen/thawed rabbit spermatozoa.     

In Vitro Evaluation of Liquid‐stored Buffalo Semen at 5°C Diluted in Soya Lecithin Based Extender (Bioxcell®), Tris‐Citric Egg Yolk,Skim Milk and Egg Yolk‐Citrate Extenders

This study was designed to compare the quality of liquid‐stored buffalo bull spermatozoa in soya lecithin based extender Bioxcell^® (BIOX), milk (MILK), tris‐citric egg yolk (TEY) and egg yolk‐citrate (EYC) extender at 5°C. Semen was collected from five Nili‐Ravi buffalo (Bubalus bubalis) bulls of 6–7 years of age with artificial vagina over a period of 3 weeks (two consecutive ejaculates once in a week). Semen ejaculates having more than 60% motility were pooled, split into four aliquots, diluted (37°C; 10 × 10⁶ motile spermatozoa/ml), cooled from 37 to 5°C in 2 h (0.275°C/min) and stored for 5 days. Sperm motility, viability, plasma membrane integrity (PMI) and normal acrosomal ridge were studied at first, third and fifth day of storage. Higher values of progressive sperm motility (%), sperm viability (%), sperm PMI (%) and normal apical ridge (%) were observed in BIOX, MILK and TEY extenders at first, third and fifth day of storage than EYC extender. Progressive sperm motility, sperm viability and sperm PMI in BIOX^® extender were not different from MILK and TEY extenders at 1st and third day storage period. However, at fifth day of storage, the values for these parameters remained significantly higher (p < 0.05) in BIOX^® compared with MILK, TEY and EYC extenders. At fifth day of storage, the semen quality parameters for Bioxcell^® were comparable to those with MILK and TEY extenders at third day of storage. In conclusion, motility, viability and PMI of buffalo bull spermatozoa remained similar in Bioxcell^®, milk and TEY extender at first and third days of storage at 5°C. Yet, the values for the aforementioned parameters in Bioxcell^® were higher compared with milk, TEY and EYC extender at fifth day of storage at 5°C.     

Characteristics of Buck Semen with Regard to Ejaculate Numbers, Collection Intervals, Diluents and Preservation Periods

To determine the number of ejaculates which can be collected within a 20‐min period after the smallest number of days of sexual rest, and a good diluent to preserve semen for routine AI, five mature Black Bengal bucks were used in three experiments. In experiment 1, semen from the bucks were collected by using artificial vagina at homosexual mounts as many times as possible during 20 min. The ejaculate numbers 1, 3 and 4 (or 5 when the buck could produce it) were examined for important semen characteristics. The mean ejaculate volume, density, mass activity, sperm motility, sperm concentrations, total spermatozoa/ejaculate, proportion of spermatozoa with normal acrosome, midpiece and tail, and the proportion with normal head morphology varied between 267 and 342 µl, 4.1–4.5 (1–5 scale), 4.1–4.2 (1–5 scale), 77–79%, 4187 × 10⁶–5064 × 10⁶/ml, 1140 × 10⁶–1746 × 10⁶, 91–94% and 99%, respectively, depending on the collection number of the ejaculate. The difference between the ejaculates was significant only with respect to volume (p < 0.05). In experiment 2, semen was collected from the bucks successively during 20 min after 1, 2, 3 and 4 day intervals, and the first ejaculates were evaluated for the above‐mentioned semen characteristics. Semen collected after 2 or more day intervals had significantly higher volume, sperm concentration and total spermatozoa/ejaculate (p < 0.05). In experiment 3, pools of two to three ejaculates were diluted (1 : 5; semen : diluent) in splits with glucose‐citrate‐egg yolk (GCEY), Tris‐fructose‐egg yolk (TFEY) or skim milk (SM) and preserved at +4 to +7°C. Before chilling or after 0 (15 min chilling), 1, 2, 3 and 4 days of preservation, semen was evaluated for motility and proportion of normal spermatozoa with respect to acrosome, midpiece and tail. In data pooled across the bucks, the sperm motility was better in GCEY and TFEY than in SM, and the proportion of normal spermatozoa was higher in SM than in the others (p < 0.05). However, the differences in proportion of normal spermatozoa between diluents were not significant when the data were analysed separately within preservation periods. The sperm motility consistently dropped after 1 day of preservation (p < 0.01); the motility remained 50% or more up to 4 days in TFEY, 3 days in GCEY and only 2 days in SM. The proportion of spermatozoa with normal acrosome, midpiece and tail, which was generally quite high ( 90%), decreased after 3 days of preservation (p < 0.01). We conclude that Black Bengal bucks can be collected three times during 20 min, every 3 days, and that buck semen holds good motility and proportion of normal spermatozoa up to 3 days in GCEY or TFEY at 4 to 7°C.     

Seminal parameters and field fertility of cryopreserved donkey jack semen after insemination of horse mares

Reasons for performing study: As mule production is often concentrated in remote areas of the world, a simplified semen cryopreservation protocol is required. Aim: To compare the seminal parameters of cryopreserved donkey semen in lactose‐EDTA and lactose‐yolk extenders and the fertility rates on horse mares. Methods: Trial 1: Sperm total and progressive motility, vigour (scale 0–5), morphology (major and minor defects) and plasma membrane integrity (HOST) were evaluated in 25 ejaculates from 5 donkey jacks immediately after collection (raw), after chilling to 5°C (chilled) and after freezing/thawing. The semen was mixed with skimmed‐milk extender, centrifuged, and then re‐suspended in lactose‐EDTA or lactose‐yolk extender. Semen was loaded into 0.5 ml straws and chilled to 5°C for 1 h, after which samples were either evaluated (chilled semen) or placed above liquid nitrogen for 20 min prior to immersion. Seminal parameters were evaluated by ANOVA and Tukey's test. Trial 2: Cryopreserved semen from 3 males was used to inseminate 53 mares at 60 oestrous cycles randomly assigned to lactose‐yolk (n = 30 cycles) or lactose‐EDTA (n = 30 cycles) extenders. Pregnancy diagnosis was performed 15 and 25 days post ovulation. The pregnancy rates were compared using Chi‐squared tests. Results: Trial 1: No significant differences were evident in any seminal parameters between extenders after either chilling or cryopreservation. Total and progressive motility were significantly (P<0.05) lower in cryopreserved semen than raw and chilled semen for both extenders. Trial 2: Pregnancy rates did not significantly differ between extenders (lactose‐EDTA extender 53.33 and 43.33%; lactose‐yolk 50.0 and 46.66% for Days 15 and 25 post ovulation, respectively). Conclusions: Cryopreservation of donkey semen using the simplified lactose‐yolk extender resulted in similar seminal parameters and fertility rates when compared to lactose‐EDTA extender. Potential relevance: Lactose‐yolk extender may be advocated as a simple, easy to prepare extender, for use in geographically isolated enterprises producing mules throughout the world.     

Soya-lecithin in extender improves the freezability and fertility of buffalo (Bubalus bubalis) bull spermatozoa

Egg yolk is routinely used as a cryoprotectant in semen extenders. However, it may contain cryoprotective antagonists, and there are hygienic risks associated with its use. Proteins of plant origin, like soya-lecithin, lack these hazards. The aim of this study was to use soya-lecithin as a cryoprotectant in extender and to investigate its effects on in vitro quality and in vivo fertility of buffalo semen. Semen from three buffalo bulls was frozen in tris-citric extender containing 5.0%, 10% or 15% soya-lecithin or 20% egg yolk. Sperm motility, plasma membrane integrity and viability were assessed post-dilution, pre-freezing and post-thaw. In Post-dilution and pre-freezing, the values for motility, plasma membrane integrity and viability remained higher (p ≤ 0.05) in extenders containing 10% soya-lecithin and control compared with extender containing 5% and 15% soya-lecithin. However, motility, plasma membrane integrity and viability were higher (p < 0.05) in extender containing 10% soya-lecithin compared with control and extenders containing 5% and 15% soya-lecithin. Semen from two buffalo bulls was frozen in tris-citric extender containing either 10% soya-lecithin or 20% egg yolk. Higher (p < 0.05) fertility rate was recorded in buffaloes inseminated with semen containing 10% soya-lecithin (56%) compared with 20% egg yolk (41.5%). The results suggest that 10% soya-lecithin in extender improves the freezability and fertility of buffalo bull spermatozoa and can be used as an alternate to egg yolk in cryopreservation of buffalo semen.     

Effects of lycopene on in vitro quality and lipid peroxidation in refrigerated and cryopreserved turkey spermatozoa

1. The effects of lycopene-enriched extenders on the in vitro quality of turkey semen including lipid peroxidation were examined after chilled and frozen storage.

2. Five pools of semen diluted in extenders containing 0, 0·05 or 0·1?mg/ml of lycopene were stored at 5°C for 48?h or cryopreserved as pellets and the following variables determined in fresh samples and samples stored chilled or frozen: sperm motility, viability, osmotic resistance, DNA integrity and lipid peroxidation (as malonaldehyde production).

3. Semen quality was generally compromised after storage, especially post-freezing. However, in the presence of the highest dose of lycopene, both the viability and osmotic-resistance of chilled spermatozoa and the DNA integrity of frozen spermatozoa were similar to those of fresh spermatozoa.

4. Greater lipid peroxidation was detected in refrigerated compared to fresh or cryopreserved spermatozoa. However, spermatozoa chilled in lycopene-enriched extenders showed significantly lower malonaldehyde levels than those chilled without lycopene, while the addition of lycopene to the freezing medium served to maintain the lipid peroxidation levels observed in fresh semen.

5. In conclusion, the presence of lycopene in the extender improved the survival of turkey spermatozoa after liquid-storage and protected DNA integrity against cryodamage. The beneficial effects of lycopene observed could be related to its capacity to diminish sperm lipid peroxidation during refrigeration or cryopreservation.     

Effect of N‐acetyl‐L‐cysteine Supplementation in Semen Extenders on Semen Quality and Reactive Oxygen Species of Chilled Canine Spermatozoa

The objective of this study was to evaluate the quality of chilled dog semen processed with extenders containing various concentrations of N‐acetyl‐L‐cysteine (NAC). Ejaculates from five dogs were collected, pooled and evaluated for concentration, motility, rapid steady forward movement (RSF‐movement), viability, acrosomal integrity and by the hypo‐osmotic swelling test (HOST). In addition, superoxide anion (O₂^‐?) production, hydroxyl radicals (OH^?) and total reactive oxygen species (tROS) were determined. The pool was divided into five aliquots, which were diluted to a final concentration of 66.66 × 10⁶ spermatozoa/ml with Tris‐glucose‐egg yolk extender containing one of the following concentrations of NAC (0, 0.5, 1, 2.5 or 5 mm ). The semen aliquots were chilled and preserved at 4°C. Semen quality was evaluated after rewarming at 72 h. Sperm motility was significantly higher with the 0.5 mm concentration compared with the control group (p = 0.001). Rapid steady forward movement was higher with the 0.5 and 1 mm concentrations compared with the control and 5 mm group (p < 0.001). Viability and HOST percentages were not significantly altered. Compared with the control, the 5 mm concentration showed significantly reduced percentages of spermatozoa with normal acrosomes (p = 0.049). None of the ROS values at 72 h were significantly affected by the presence of NAC in semen extenders, although all NAC concentrations showed lower O₂^‐? and OH^? values compared with the control. Only the concentrations of 1 and 5 mm inhibited the significant increase of tROS values after 72 h, compared with the fresh semen value. In conclusion, NAC supplementation of semen extenders is beneficial to semen motility of canine spermatozoa during chilling with the 0.5 mm concentration being the most effective, although no significant ROS inhibition was observed at 72 h.     

Effect of Oviductal Proteins on Structural and Functional Characteristics of Cryopreserved Sperm in Murrah Buffaloes*

The present study was undertaken to elucidate the effect of non‐luteal oviductal proteins on sperm characteristics in Murrah buffaloes. Oviducts from healthy buffaloes were collected immediately after slaughter and the oestrous cycle phase was determined as either luteal or non‐luteal based on ovarian morphology. Non‐luteal oviducts (n = 80) were flushed from the isthmic end of the oviduct with PBS, fluid was centrifuged at 10 000 g at 4°C for 20 min and then dialysed and clarified. The supernatant obtained was lyophilized to concentrate the protein and stored at ?20°C till use. Sixteen good quality ejaculates from four Murrah buffalo bulls were collected using an artificial vagina. After fresh semen analysis, each ejaculate was split into two parts and extended in Tris–citrate–egg yolk glycerol dilutor. Part I of the split ejaculate was treated with non‐luteal oviductal proteins at the dose rate of 1 mg/ml of diluted semen, while part II remained as control. The extended semen was equilibrated for 4 h at 5°C, filled in 0.5 ml French straws, exposed to LN₂ vapour, plunged into LN₂ and then stored at ?196°C. The equilibrated and frozen–thawed semen was evaluated for sperm motility, viability, acrosomal integrity, cervical mucus penetration test and hypo‐osmotic sperm swelling test (HOST). In frozen–thawed semen, the percentage of sperm motility, viability and acrosomal integrity was significantly (p < 0.05) higher in the treatment group compared to the control group. The incorporation of non‐luteal oviductal proteins in the extender increased the ability of sperm to penetrate cervical mucus both after equilibration and the freeze‐thaw process. Similarly, the proportion of sperm with intact plasma membrane, as revealed by HOST values, was also significantly (p < 0.05) higher in the treatment group (32.6%) than the control group (27%) in frozen–thawed semen. It was inferred that incorporation of non‐luteal whole oviductal fluid proteins improved the sperm quality in frozen–thawed semen in Murrah buffaloes.     

Optimization of Ram Semen Cryopreservation Using a Chemically Defined Soybean Lecithin‐Based Extender

The purpose of the present study was to investigate the effects of a chemically defined soybean lecithin‐based semen extender as a substitute for egg yolk‐based extenders in ram semen cryopreservation. In this study, 28 ejaculates were collected from four Zandi rams in the breeding season and then pooled together. The pooled semen was divided into six equal aliquots and diluted with six different extenders: (i) Tris‐based extender (TE) containing 0.5% (w/v) soybean lecithin (SL0.5), (ii) TE containing 1% (w/v) soybean lecithin (SL1), (iii) TE containing 1.5% (w/v) soybean lecithin (SL1.5), (iv) TE containing 2% (w/v) soybean lecithin (SL2), (v) TE containing 2.5% (w/v) soybean lecithin (SL2.5) and (vi) TE containing 20% (v/v) egg yolk (EYT). After thawing, sperm motility and motion parameters, plasma membrane and acrosome integrity, apoptosis status and mitochondrial activity were evaluated. The results shown that total and progressive motility (54.43 ± 1.33% and 25.43 ± 0.96%, respectively) were significantly higher in SL1.5 when compared to other semen extenders. Sperm motion parameters (VAP, VSL, VCL, ALH and STR) were significantly higher in SL1.5 compared to other extender, with the exception of SL1 extender. Plasma membrane integrity (48.86 ± 1.38%) was significantly higher in SL1.5 when compared to other semen extenders. Also, percentage of spermatozoa with intact acrosome in SL1.5 (85.35 ± 2.19%) extender was significantly higher than that in SL0.5, SL2.5 and EYT extenders. The results showed that the proportion of live post‐thawed sperm was significantly increased in SL1.5 extender compared to SL0.5, SL2 and EYT extenders. In addition, SL1, SL1.5 and SL2.5 extenders resulted in significantly lower percentage of early‐apoptotic sperm than that in EYT extender. There were no significant differences in different semen extenders for percentage of post‐thawed necrotic and late‐apoptotic spermatozoa. Also, the results indicated that there are slight differences for percentage of live spermatozoa with active mitochondria between extenders. In conclusion, SL1.5 extender was better than other extenders in most in vitro evaluated sperm parameters.     

Effects of Thawing Temperature and Post‐thaw Dilution on the Quality of Cat Spermatozoa

The present study aimed to compare cat sperm quality after thawing using two different temperatures (37 and 70°C) and to investigate the effects of post‐thaw dilution on the sperm quality and longevity of ejaculated cat spermatozoa. Six ejaculates of each of six male cats were collected using an electroejaculator (total 36 ejaculates). The semen was frozen in 0.25‐ml straws using a Tris egg yolk extender containing Equex STM paste. Four straws prepared from each ejaculate were thawed at four different occasions; (i) at 37°C for 15 s, (ii) at 37°C for 15 s and diluted 1 : 2 with Tris buffer (v/v), (iii) at 70°C for 6 s, (iv) at 70°C for 6 s and diluted 1 : 2 with Tris buffer (v/v). The percentages of motile spermatozoa, the scores of progressive motility, the percentages of spermatozoa with intact plasma membrane (using SYBR‐14/EthD‐1 stains) and intact acrosome (using fluorescein isothiocyanate conjugated peanut agglutinin/propidium iodide stains) were evaluated in fresh semen at 0, 2, 4 and 6 h after thawing. The thawing temperature had no effect on any sperm parameters throughout the incubation period (p > 0.05). The dilution after thawing improved sperm motility, progressive motility and acrosome integrity (p < 0.05). The thawing of cat spermatozoa and subsequently diluting with Tris buffer resulted in an immediate (at 0 h) overall (combined over temperature) percentage of motile sperm of 64.8 ± 10.7 (mean ± SD), a score of progressive motility of 4.0 ± 0.5, a percentage of spermatozoa with intact plasma membrane of 64.4 ± 12.1 and intact acrosome of 44.8 ± 20.2. In conclusion, frozen cat semen can be thawed either at 37 or 70°C and post‐thaw dilution is recommended to reduce the toxic effect of some ingredients in the extender during post‐thaw incubation.