Testing Usability of Butylated Hydroxytoluene in Conservation of Goat Semen

The objective of this study was to investigate whether butylated hydroxytoluene (BHT) could be used as a suitable supporter or alternative of egg yolk during preservation of goat spermatozoa. Three in vitro experiments and a fertility test were conducted to evaluate the effect of BHT on viability of chilled‐stored semen as well as motility and kidding rate of frozen‐thawed spermatozoa. In the first two experiments, ejaculates (n = 30/experiment) were collected from 10 bucks, split, diluted with egg yolk‐based and egg yolk‐free extenders supplemented with or without 0.3, 0.6, 2, 5 and 8 mm BHT and stored at 5°C for 168 h. In the third experiment, 30 ejaculates were collected from the above‐mentioned bucks, split and diluted with egg yolk‐free extenders supplemented with or without 0.3, 0.6 and 0.9 mm BHT and egg yolk‐based extenders supplemented with or without 5 mm BHT. Diluted semen was cooled to 5°C over a period of 4 h, frozen and thawed in the form of 0.3‐ml pellets. In the fertility test, 75 ejaculates were collected from two proven fertile bucks, split, diluted with egg yolk‐free extenders containing 0.6 mm BHT and egg yolk‐based extenders supplemented with or without 5 mm BHT, frozen and thawed as described above. An insemination volume of 0.6 ml containing 120–140 × 10⁶ progressively motile spermatozoa was used for a single cervical insemination of cloprostenol‐synchronized does (n = 230). The results showed that addition of 5 mm BHT to egg yolk‐deficient (2.5%) extenders significantly improved viability of chilled‐stored semen together with motility (48.5%) and fertility (62.5%) of frozen‐thawed spermatozoa. Replacement of egg yolk in semen extenders by 0.6 mm BHT could sustain not only viability of chilled‐stored semen but also post‐thaw motility (47.5%) and fertility (53.75%) of frozen‐thawed spermatozoa. In conclusion, supplementation of semen diluents with BHT can ameliorate preservability of goat sperm.     

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.     

The Effect of Sperm Concentration and Storage Vessel on Quercetin‐Supplemented Rabbit Semen During Chilled Storage

Extending the shelf life of chilled rabbit spermatozoa is vital for the expansion of the farmed rabbit industry. This study evaluated the relationship between sperm concentration and packaging on in vitro quality of chilled rabbit semen over 96 h. Semen was collected from adult bucks (n = 4) and pooled at 37°C following evaluation. Pooled ejaculates were diluted with a Tris‐based extender supplemented with 100 μm quercetin to a concentration of 15, 30 or 60 × 10⁶ spermatozoa/ml, packaged into plastic tubes or 0.5‐ml straws and stored at 15°C. Sperm quality was assessed by computer‐assisted sperm Analysis [total motility (tMOT)] and flow cytometry [viability, acrosome integrity, H₂O₂ production, plasma membrane disorder, apoptosis and DNA fragmentation index (DFI)] at 0, 48, 72 and 96 h. From 48 h, concentrations of 30 and 60 × 10⁶ spermatozoa/ml reported the highest tMOT, irrespective of storage vessel (p < 0.05). Storage in straws reduced oxidative stress and improved plasma membrane stability. The %DFI, mean DFI and SD‐DFI were increased in spermatozoa stored in tubes compared with straws (p < 0.05). Although the use of low sperm concentrations in artificial insemination doses would facilitate greater dispersion of genetically superior rabbit bucks, dilution to 15 × 10⁶ spermatozoa/ml had a detrimental impact on motility. As such, chilled storage at 30 × 10⁶ spermatozoa/ml may provide a suitable balance between motility and H₂O₂ production to best maintain overall sperm function and should be evaluated in a large‐scale AI trial.     

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.     

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.     

Glycerol,Methyl‐Formamide and Dimethyl‐Formamide in Canine Semen Cryopreservation

The aim of the present work was to compare the efficiency of methyl‐formamide (MF), dimethyl‐formamide (DF) and glycerol (GL) as cryoprotectants in canine semen cryopreservation. For the experiment, pooled semen was submitted to one of the three cryoprotectants, with a final concentration of 3% in egg yolk–TRIS extender. Semen was subjectively evaluated for total and progressive motility, vigour and morphology. Sperm membrane functional integrity was assessed by hypo‐osmotic swelling test (HOST), and longevity was assessed using the thermoresistance test (TRT). Fresh semen showed normal physical and morphological characteristics. After thawing, differences were observed between semen frozen using GL and DF, regarding total and progressive motility and vigour (p < 0.05), but not between MF and GL or MF and DF. Means for total motility, progressive motility, vigour and morphologically normal spermatozoa were, respectively, 69.0 ± 5.4%, 61.0 ± 7.4%, 2.9 ± 0.5 and 57.1 ± 5.0% for GL; 59.0 ± 8.9%, 50.0 ± 10.0%, 2.5 ± 0.7 and 66.9 ± 7.7% for MF; and 44.0 ± 21.0%, 37.0 ± 19.8%, 2.1 ± 0.6 and 61.1 ± 5.5% for DF. On HOST, GL was superior (p < 0.05) to MF and DF (57.8 ± 12.4%, 35.8 ± 18.4% and 34.4 ± 9.4%, respectively). During the TRT, both GL and MF were superior to DF, with no differences between GL and MF. In conclusion, the use of MF as cryoprotectant showed results similar to GL, and can be considered as an alternative in canine semen cryopreservation. Further studies testing different concentrations of MF may improve its effects on cryopreservation of canine semen.     

添加褪黑素对牛精液品质的影响

为探讨褪黑素(melatonin,MLT)在牛精液保存中的抗氧化作用,本研究利用目测法、低渗膨胀法和考马斯亮兰染色法分析了不同MLT浓度(0、10^-3、10^-4和10^-5 mol/L)和不同孵育时间(1、4和8 h)对精子活力、质膜和顶体完整性的影响。结果发现,与对照组相比,在稀释液中分别添加10^-3和10^-4 mol/L MLT均可显著提高精子活力、质膜完整率和顶体完整率(P＜0.05),且10^-3 mol/L MLT添加效果最佳;MLT(10^-3 mol/L)组在27 ℃孵育1、4 h后,其精子的活力、质膜完整率和顶体完整率均显著高于对照组(P＜0.05)。因此,添加10^-3 mol/L MLT可明显改善牛精液品质,提高牛精液的保存时间和保存质量。     

Investigation of in vitro measurable sperm attributes and their influence on electroejaculated bull semen with a fixed‐time artificial insemination protocol in Australian Bos indicus cattle

Increasing use of fixed‐time artificial insemination (FTAI) in beef cattle production has presented an opportunity for the use of fresh or chilled semen as an alternative to standard cryopreserved semen. The objective of this study was to examine in vitro sperm function and pregnancy rate of electroejaculated semen, chilled and stored for 48 hr, compared to conventionally cryopreserved semen with an optimized FTAI protocol in Brahman cattle. Semen from three Brahman bulls was collected, and aliquots were extended in either chilled (at 5°C) or frozen (LN₂) in a Tris‐egg yolk extender base with 2.4% or 7.0% glycerol, respectively. Semen samples were assessed 48 hr after collection or post‐thaw and warming, for sperm motility, in vitro sperm function and fertilizing ability, and used in a FTAI programme. The overall pregnancy rates was significantly different (p < .01) after FTAI with frozen (n = 173; 53.2%) and chilled semen (n = 174; 31.6%). In contrast, the in vitro sperm assessment showed that the chilled semen had significantly faster motility (p < .05), a higher proportion of progressively motile spermatozoa (p < .05), with significantly higher proportions of acrosome intact, viable spermatozoa (p < .01). This study showed that reasonable pregnancy rates in Brahman cattle can be achieved using FTAI with chilled semen collected using electroejaculation and stored for up to 48 hr. However, improvements in semen extenders are required in consideration of semen collection method to improve the longevity of sperm fertilizing ability to significantly increase FTAI output using chilled storage of bull semen.     

Cooled Storage of Canine Semen: in vitro Effects of Different Concentrations of an Antibiotic Combination on Growth of Mollicutes

The aim of this study was to examine effects of an antibiotic combination at different concentrations on growth of mycoplasma and ureaplasma during cooled storage of canine semen (n = 20). Semen aliquots were diluted with Tris–citric acid–fructose–egg yolk extender containing either 1.0 g/l streptomycin and 0.6 g/l benzylpenicillin (control) or a combination of gentamycin, tylosin, lincomycin and spectinomycin (GTLS‐1: 0.25, 0.05, 0.15 and 0.3; GTLS‐2: 0.5, 0.1, 0.3 and 0.6; GTLS‐3: 1.0, 0.2, 0.6 and 1.2 g/l). Samples were assessed for motility and membrane integrity by computer‐assisted sperm analysis immediately after dilution and at 24, 48 and 72 h of cooled storage. Morphologically, normal spermatozoa were determined, and bacterial culture was performed at 24 and 72 h. Mycoplasma spp. were detected in 14 of 20 ejaculates (70%) with severe growth in 12 samples. A reduction but not total elimination of mycoplasma growth occurred in all GTLS extenders with the most pronounced reduction in group GTLS‐3 (control vs GTLS‐1 and GTLS‐2 p < 0.05, control vs GTLS‐3 p < 0.001). Ureaplasmas were detected in four ejaculates, and growth was reduced to the same extent in GTLS and control extender. Progressive motility in all groups, total motility in groups GTLS 1–3 and percentage of membrane‐intact spermatozoa in groups GTLS 2 and 3 decreased slightly (p < 0.05) over time. In conclusion, dilution of canine semen with GTLS extender has no major detrimental effects on spermatozoa during cooled storage. It reduced the growth but did not totally eliminate mycoplasmas and ureaplasmas from cooled‐stored dog semen.     

Freezability of Tushin Ram Semen Extended with Goat or Cow Milk Based Extenders

Cow milk is used as an extender for ram semen cryopreservation. Caseins, the major proteins of milk, appear to provide some protective effect to sperm during cryopreservation. Goat milk has unique casein structure. The aim of this study was to investigate effect of goat milk, as a main semen extender, on freezability of Tushin Ram semen. For this aim, ejaculates from four Tushin rams were collected with artificial vagina and pooled. Pooled semen was separately extended with four different extenders: TRIS based (TRIS), cow skim milk based (CSM) (10 g/100 ml), cow semi‐skim milk based (CSSM) and goat semi‐skim milk based (GSSM) extenders, containing egg yolk and glycerol. The semen was cryopreserved and stored in liquid nitrogen until examination date. After thawing (at 37°C for 1 min), sperm motility, viability, morphology, acrosome and membrane integrity (HOST) were evaluated. Although, there was not any significant differences between extenders in post‐thaw percentage of viable spermatozoa (p > 0.05), Tushin ram semen extended with GSSM or CSM extenders had significantly higher post‐thaw percentage of progressive motility (25.0% and 30.8% respectively), compared with CSSM and TRIS (7.5% and 14.1% respectively, p < 0.001). Moreover, lowest abnormality percentage of post‐thaw spermatozoa were detected in ram semen extended with GSSM (49.5%) and CSM (51.5%), compared with CSSM (65.7%) and TRIS (60.7%) (p < 0.05). Whilst the results were considered, it was concluded that goat milk based extenders may be effectively and trustfully used in cryopreservation of Tushin ram semen, instead of cow milk and Tris based extenders, as a main extender.     

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.     

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.     

Functional Sperm Parameters and Fertility of Bull Semen Extended in Biociphos-Plus® and Triladyl®

Twenty ejaculates from five dairy AI‐bulls were used to compare, in a split‐sample experiment, the fertility [56 day‐non‐return‐rate (NRR) from more than 14000 AI) and sperm viability post‐thaw of semen diluted with an egg yolk‐ (Triladyl®) or soybean‐based (Biociphos‐Plus®) commercial extender. The in vitro evaluations were divided in two experiments. Experiment 1 (n = 20) included post‐thaw evaluations of motility (subjective and computerized), membrane integrity (CalceinAM/EthD‐1, SYBR‐14/PI, and osmotic resistance test; ORT), and capacitation status (CTC/EthD‐1). Experiment 2 (n = 10) included evaluations of the capacitation‐(CTC/EthD‐1) and acrosome status (FITC‐PSA/EthD‐1) during incubation with/without a challenge with solubilized zona pellucida proteins (SZP). No significant difference in the fertility (69.1 ± 0.8 versus 69.2 ± 0.8) results was found between the two extenders. In experiment 1, the computerized motility evaluations post‐thaw (CASA) showed higher values for Biociphos‐Plus® processed semen for the velocity patterns and lateral sperm head displacement. After 6 h at room temperature (20–22°C) all the CASA motility patterns were significantly higher for Biociphos‐Plus®. The proportion of spermatozoa with intact membranes assessed by CalceinAM was significantly higher in Biociphos‐Plus® (p < 0.001) compared to Triladyl®, but such difference was not seen when using SYBR‐14 or the ORT‐assay. When using the CTC/EthD‐1 assay, a lower proportion of acrosome reacted (AR) spermatozoa post‐thaw (p < 0.01) was found in Biociphos‐Plus® processed semen, as well as a tendency (p < 0.07) for a higher number of uncapacitated spermatozoa. In experiment 2, the proportion of uncapacitated spermatozoa was significantly higher for Biociphos‐Plus® when semen was incubated (38°C and 5% CO₂) without SZP at both 0 (p < 0.001) and 30 min (p < 0.05). Concomitantly, Triladyl® showed a higher percentage of capacitated spermatozoa at 0 (p < 0.01), 30 (p < 0.05) and 120 min (p < 0.05). A higher (p < 0.05) incidence of AR‐spermatozoa was seen in Triladyl® at the beginning of the incubation with SZP. No significant difference between extenders was detected for the acrosome status by the FITC‐PSA‐assay. Incubation with SZP induced acrosome reaction of capacitated spermatozoa in both extenders, which was detected by CTC and FITC‐PSA assays. In conclusion, fertility was not affected by Biociphos‐Plus® when 15 × 10⁶ of spermatozoa per AI dose were inseminated. The finding that higher frequencies of spermatozoa seemed more membrane stable post‐thaw, when frozen in Biociphos‐Plus®, might indicate that this extender better protects the sperm viability compared with Triladyl®.     

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.     

Association between Myeloperoxidase Concentration in Equine Frozen Semen and Post‐Thawing Parameters

Despite improvement of techniques, semen of 20% of stallions remains unfreezable. Recent studies focused on the impact of reactive oxygen species and oxidant enzymes on semen characteristics. Myeloperoxidase (MPO) is a pro‐oxidant enzyme contained in and released by neutrophils during degranulation or after cell lysis. It is responsible for the formation of hypochlorous acid, a strong oxidant agent, which could damage spermatozoa. The aim of this study was to determine the relation between MPO concentration and characteristics of frozen semen from stallions. Thirty‐five straws from different stallions were analysed. Post‐thawing spermatozoal concentration, and progressive and total motility were determined by Computer‐Assisted Semen Analysis. Freezability was determined according to post‐thawing progressive motility (above or below 15%). Percentage of alive spermatozoa and abnormal forms was determined after Eosin–Nigrosin and Diff‐Quick^® staining, respectively. Post‐thawing MPO concentration was measured by enzyme‐linked immunosorbent assay (ELISA). Our study shows that frozen thawed semen contains large amounts of free MPO. We also observed that post‐thawing MPO ELISA assay can be used as an indicator of equine semen freezability. High MPO concentration samples showed lower total and progressive motility. A higher proportion of abnormal head shape associated with acrosome reaction was observed in our late examinations of the high concentration MPO group. Our results show that MPO adversely affects total and progressive motility of equine semen. A negative correlation between normal motile forms and MPO concentration was also observed. The effect of MPO on dead or abnormal forms remains to be precised.     

Cryopreservation of Ram Semen in Extenders Containing Soybean Lecithin as Cryoprotectant and Hyaluronic Acid as Antioxidant

A soybean lecithin‐based extender supplemented with hyaluronic acid (HA) was assayed for effectiveness to improve the quality of frozen–thawed ram semen. HA has not been tested yet in an extender containing soybean lecithin for freezing ram semen. Thus, the aim of this study was to analyse the effects of soybean lecithin at 1% or 1.5% along with HA at 0, 0.5 and 1 mg ml^‐1 in a Tris‐based extender on the motion characteristics, membrane integrity (HOST), viability, GSH peroxidase (GSH‐PX) activity, lipid peroxidation and acrosomal status after freezing–thawing. Semen was collected from four Mehraban rams during the breeding season and frozen in the six lecithin×HA extenders. The extender containing 1.5% lecithin supplemented with no HA yielded higher total motility (52.5%±1.6), viability (55.8%±1.6) and membrane integrity (44.5%±1.7), but the effects of the lecithin concentration did not reach signification. Linearity‐related parameters, ALH, BCF, lipid peroxidation, GSH‐PX activity, morphology and acrosomal status were not affected by the extender composition. In general, adding HA significantly decreased sperm velocity (1 mg ml^‐1 HA), total motility (only with 1.5% lecithin), viability (1 mg ml^‐1 HA for 1% lecithin; both concentrations for 1.5% lecithin) and membrane integrity. In conclusion, adding HA to the freezing extender supplemented with soybean lecithin failed to improve quality‐related variables in ram semen. Increasing the lecithin content could have a positive effect, but further studies are needed.     

Arachidic Acid in Extender Improves Post‐thaw Parameters of Cryopreserved Nili‐Ravi Buffalo Bull Semen

Cryopreservation process reduces lipids and phospholipids from buffalo bull spermatozoa. It was therefore hypothesized that supplementation of fatty acid to extender may improve the post‐thaw quality of buffalo semen. The objective was to evaluate the effect of arachidic acid supplementation in extender on post‐thaw quality of buffalo bull (Bubalus bubalis) spermatozoa. Semen was collected from three adult Nili‐Ravi buffalo bulls of similar age group with artificial vagina (42°C) for 3 weeks (replicate). Qualified semen ejaculates (n = 18) were split into four aliquots and diluted in tris–citric acid extender containing 0.0 (control), 5.0, 10.0 and 20.0 ng/ml at 37°C having approximately 50 × 10⁶ spermatozoa/ml. Diluted semen was cooled to 4°C in 2 h and equilibrated for 4 h at 4°C. Cooled semen was filled in 0.5‐ml straws at 4°C, kept on liquid nitrogen vapours for 10 min and plunged in liquid nitrogen for storage. Thawing of frozen semen was performed after 24 h at 37°C for 30 s. Sperm progressive motility (%) was improved in a dose‐dependent manner by supplementing arachidic acid at 5.0, 10.0 and 20.0 ng/ml compared with control. Structural and functional integrity of sperm plasma membrane (%), number of acrosome‐intact live sperm (%) and sperm chromatin integrity (%) were better (p < 0.05) in extender having 5.0 ng/ml of arachidic acid compared with control. At 10.0 ng/ml, these values did not vary (p > 0.05) from those at 5.0 ng/ml. Further improvement in structural and functional integrity of sperm plasma membrane, number of acrosome‐intact live sperm and chromatin integrity was observed at 20.0 ng/ml of arachidic acid in extender. In conclusion, arachidic acid supplementation in extender improved the post‐thaw quality parameters of cryopreserved Nili‐Ravi buffalo bull spermatozoa. Among the arachidic acid concentrations studied, maximum improvement in post‐thaw semen quality parameters was observed at 20.0 ng/ml.     

Tris-Lecithin Extender Supplemented With Antioxidant Catalase for Chilling of Canine Semen

The aims were to evaluate the suitability of a non-commercial TRIS-lecithin (LC) extender and the effect of different concentrations of catalase (CAT) on motility, capacitation status (Chlortetracycline-assay) and zona pellucida (ZP) binding capacity of canine spermatozoa stored at +5°C for 4 days. The sperm-rich fractions of the ejaculates of four stud dogs were divided into four aliquots. After centrifugation, sperm pellets were diluted (200 × 10⁶ sperm/ml) in TRIS buffer, citric acid, fructose, antibiotics, supplemented with 20% egg yolk (TRIS-EY) or 0.04% soybean lecithin (TRIS-LC) with CAT (150 or 450 UI/ml) or without CAT, and then preserved at 5°C for 4 days. The results showed that LC is a valid alternative to EY for chilling canine semen, as similar rates of motility, number of uncapacitated spermatozoa and of spermatozoa binding the oocyte ZP were obtained in semen chilled in TRIS-LC or TRIS-EY. Different concentrations of CAT in a TRIS-LC based extender did not improve the quality of semen after chilling. However, a concentration of 150 UI/ml CAT resulted in an increased number of spermatozoa bound to the oocyte ZP, after 4 days of chilling when compared to semen chilled with TRIS-EY and TRIS-LC. In conclusion, an animal protein-free extender with soybean LC, as a replacement of EY, is suitable for 4 days chilling of canine spermatozoa, but the addition of CAT does not improve general semen quality except for a slight effect on sperm-ZP binding.     

The benefits of cooling boar semen in long‐term extenders prior to cryopreservation on sperm quality characteristics

This study investigated the effects of long‐term extenders on post‐thaw sperm quality characteristics following different holding times (HT) of boar semen at 17 and 10°C. Sperm‐rich fractions, collected from five boars, were diluted in Androhep^® Plus (AHP), Androstar^® Plus (ASP), Safecell^® Plus and TRIXcell^® Plus (TCP) extenders. The extended semen samples were held for 2 hr at 17°C (HT 1) and additionally for 24 hr at 10°C (HT 2), after they were evaluated and frozen. CASA sperm motility and motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome integrity were assessed in the pre‐freeze and frozen‐thawed semen. The Vybrant Apoptosis Assay Kit was used to analyse the proportions of viable and plasma membrane apoptotic‐like changes in spermatozoa. Results indicated that boar variability, extender and HT significantly affected the sperm quality characteristics, particularly after freezing‐thawing. Differences in the pre‐freeze semen were more marked in the sperm motion patterns between the HTs. Pre‐freeze semen in HT 2 showed significantly higher VCL and VAP, whereas no marked effects were observed in the sperm membrane integrity and viability (YO‐PRO‐1^?/PI^?) among the extenders. Post‐thaw sperm TMOT and PMOT were significantly higher in the AHP and ASP extenders of HT 2 group, whereas VSL, VCL and VAP were markedly lower in the TCP extender. Furthermore, spermatozoa from the AHP‐ and ASP‐extended semen of HT 2 group were characterized by higher MMP, PMI and NAR acrosome integrity following freezing‐thawing. In most of the extenders, the incidence of frozen‐thawed spermatozoa with apoptotic‐like changes was greater in HT 1. The findings of this study indicate that holding of boar semen at 10°C for 24 hr in long‐term preservation extenders modulates post‐thaw sperm quality characteristics in an extender‐dependent manner. These results will further contribute to the improvement in the cryopreservation technology of boar semen.     

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.