Melatonin improves post-thaw sperm quality after mild testicular heat stress in rams.

The objective was to determine effects of slow-release melatonin on post-thaw sperm quality in rams exposed to mild testicular heat stress (HS; scrotal neck insulation). Twelve yearling Dorset rams were randomly and equally allocated to receive either 36 mg melatonin in 1 ml corn oil or 1 ml corn oil injected subcutaneously (SQ); 15 day later, all rams had HS for 96 h (start of HS = start of Week 0). Semen was collected before HS and once weekly from Weeks 1 to 7, extended in Steridyl CSS One Step, held at 5°C for ~3 h, loaded into 0.5 ml straws, held 5 cm above liquid nitrogen for 10 min and then plunged. Computer assisted semen analysis (CASA) was conducted on frozen-thawed sperm. There were group and week effects for total and progressive motility (p < .001), plus group and week effects and group*week interactions (p < .001) for post-thaw total abnormalities, acrosome integrity, post-thaw sperm DNA fragmentation index (DFI) and high mitochondrial membrane potential (HMMP). Post-thaw sperm total and progressive motility, acrosome integrity and HMMP were higher (p < .05) in melatonin versus control groups from Weeks 1 to 7, and the melatonin group reached baseline level (pre-heat stress) at Week 7 (75.79 ± 0.96, 65.48 ± 1.51, 75.00 ± 0.89 and 67.00 ± 1.06, respectively; mean ± SEM). Conversely, post-thaw sperm total abnormalities and DFI were lower (p < .05) in melatonin versus control, and both reached baseline at Week 7 in the melatonin group (26.00 ± 0.57 and 5.66 ± 0.17, respectively). Coiled tails, distal midpiece reflexes, distal cytoplasmic droplets, ruffled acrosomes, bowed midpieces, pyriform heads and knobbed acrosomes were the most common abnormalities in both groups, with lower percentages in melatonin-treated rams. Results supported our hypothesis that HS reduces post-thaw sperm quality, and that melatonin lessens those reductions, manifested by significantly better total and progressive motility, acrosome integrity and HMMP, and fewer sperm total abnormalities and DFI.

Effects of reduced glutathione supplementation in semen freezing extender on frozen-thawed bull semen and in vitro fertilization.

During cryopreservation, spermatozoa may suffer cold and cryo-induced injuries -associated with alterations in cell defense systems- that are detrimental to their function and subsequent fertility. This study aimed to determine the efficacy of supplementing the semen freezing extender with the antioxidant reduced glutathione (GSH) in cattle. Semen was collected from four bulls and diluted in a freezing extender supplemented with or without GSH (0, 1, 5, and 10 mM) before the cooling step of the cryopreservation process. After thawing, the quality of the frozen-thawed semen was investigated for motility, viability, acrosomal and DNA integrity, and subsequent embryo development after in vitro fertilization of bovine oocytes. Additionally, semen from one of the bulls was used to analyze semen antioxidative potential, sperm penetration into oocytes, male pronucleus formation rate, and embryo DNA integrity. The sperm quality varied among bulls after GSH supplementation. One bull had decreased sperm total motility, and two bulls had decreased sperm DNA integrity. GSH supplementation had positive effects on embryo development for three bulls. Two of them showed both improved cleavage and blastocyst formation rates, while the other one only showed an improved cleavage rate. We observed positive effects on early male pronucleus formation and no negative effects on DNA integrity and cell number in blastocyst stage embryos. Although the effect varies depending on individual bulls and GSH concentration, GSH supplementation in semen may improve in vitro embryo production from frozen semen.

Fertility response of artificial insemination methods in sheep with fresh and frozen-thawed semen.

The aim of this study was to evaluate the fertility response of artificial insemination (AI) methods with fresh and frozen sperm in sheep. In experiment 1, one hundred and fifty fat tailed Zandi ewes were assigned into 3 equal groups and inseminated with three AI methods consisting of vaginal, laparoscopic and trans-cervical AI with fresh semen. In experiment 2, a factorial study (3 AI methods × 2 extenders) was used to analyze the effects of three AI methods and two freezing extenders containing soybean lecithin (SL) or Egg yolk (EY) on reproductive performance of 300 fat tailed Zandi ewes. Also, total motility, progressive motility, viability and lipid peroxidation of semen were evaluated after freeze-thawing in two extenders. In result, there was no significant difference among three AI methods when fresh semen was used. In experiment 2, the highest percentage of pregnancy rate, parturition rate and lambing rate were obtained in laparoscopic AI group (P < 0.05). Although pregnancy rate, parturition rate and lambing rate in trans-cervical group were higher (P < 0.05) than vaginal group, the results were not as high as laparoscopic group. No difference was observed between SL and EY extenders and their performance was close to each other. It can be concluded that although no difference was observed on reproductive performance for fresh semen, trans-cervical AI was more efficient than vaginal method when frozen-thawed semen was used, but its efficiency was not as high as laparoscopic method. Also, SL extender can be an efficient alternative extender to preserve ram sperm during cryopreservation procedure without adverse effects of EY.

Sperm characteristics and heterologous in vitro fertilisation capacity of Iberian ibex (Capra pyrenaica) epididymal sperm, frozen in the presence of the enzymatic antioxidant catalase.

The aim of this work was to evaluate the protective effect of catalase (CAT) on frozen/thawed ibex epididymal sperm recovered post mortem, and to detect any harmful effect this might have on sperm fertilisation capacity. Epididymal spermatozoa were diluted using a Tris-citric acid-glucose medium (TCG) composed of 3.8% Tris (w/v), 2.2% citric acid (w/v), 0.6% glucose (w/v), 5% glycerol (v/v), and 6% egg yolk (v/v). Sperm masses from the right epididymis were diluted with TCG medium, while those from the left were diluted with TCG medium supplemented with 200IU/mL CAT. Heterologous in vitro fertilisation (IVF) was used to assess the fertilisation capacity of this sperm. The addition of CAT to the extender did not improve frozen/thawed sperm variables. Moreover, a reduced fertilisation capacity was detected: sperm diluted with TCG provided 25.5% 2PN zygotes, while just 13.2% was recorded for that diluted with TCG-CAT (P<0.01). The percentage of cleaved embryos at 48hpi was higher (P<0.01) with the TCG sperm than with the TCG-CAT sperm (16.7% vs. 7.6%). The use of 200IU/mL CAT as an additive cannot, therefore, be recommended for the preservation of ibex epididymal sperm. Other antioxidants should, however, be tested in both this and related wild mountain ungulates.

Factors affecting the analysis and interpretation of sperm quality in frozen/thawed stallion semen.

In the current study, we examined: 1) the agreement (bias) between fluorescence-based methods (NucleoCounter-SP100 [NC] vs. flow cytometry [FC]) for determining the viability (VIAB) of frozen/thawed stallion sperm; 2) the agreement between post-thaw sperm total motility (TMOT) and VIAB; 3) whether a difference between TMOT and VIAB [VIAB - TMOT] in frozen/thawed stallion sperm could be explained by the level of lipid peroxidation in viable sperm (VLPP); 4) the repeatability of post-thaw analysis of sperm quality; and 5) the effect of final post-thaw semen dilution (10, 30, or 50 x 106 sperm/mL) on sperm motion characteristics. Post-thaw VIAB was similar between NC and FC (P > 0.05), and the agreement between these two methods was high (bias: 1 to -3). The agreement between post-thaw TMOT and VIAB decreased as the pre-freeze percentages of morphologically normal sperm and DNA quality decreased: bias - 4 to - 25. The bias between [VIAB - TMOT] and VLPP ranged from - 5 to 7. Differences in post-thaw sperm quality (TMOT, PMOT, VIAB, and sperm concentration) were not observed when analyzing one or three straws per ejaculate (P > 0.05). There was no effect of post-thaw sperm concentration (i.e., 10 vs. 30 vs. 50 x 106 sperm/mL) on sperm motion characteristics (P > 0.05). This study reports factors other than post-thaw sperm motility that warrant further consideration when analyzing frozen/thawed stallion sperm.

The Effects of Ferulic Acid, Tryptophan, and L-Glutamine on the Cryopreservation of Mouse Spermatozoa.

In this study, the effects of ferulic acid (0.1, 1, ve 10 mM), tryptophan (5, 25, ve 50 mM), and L-glutamine (10, 50, ve 100 mM) at different doses added to 18% raffinose + 3% skimmed milk powder sperm extender on the freezing of mouse spermatozoa in liquid nitrogen were investigated. The combination of 18% raffinose + 3% skimmed milk powder without additives was used as the control group. Frozen spermatozoa were thawed in a 37°C water bath for 30 seconds. After freeze-thawing, motility, dead spermatozoa ratio, plasma membrane integrity, abnormal acrosome ratio, motility endurance (for 4 hours), and cell apoptosis tests were performed in Human Tubal Fluid (HTF). Compared with the control group after freezing and thawing, the highest motility and plasma membrane integrity were obtained in the 10 mM L-glutamine group with 56.6% ± 2.11% and 77.8% ± 0.87%, respectively (p < 0.05). In addition, when compared to the control group, the lowest rate of dead spermatozoa and abnormal acrosome was found in the 10 mM L-glutamine group as 26.0% ± 1.46% and 6.3% ± 1.09%, respectively (p < 0.05). The highest motility values for spermatozoa endurance were determined in the 10 and 50 mM L-glutamine groups up to the 4th hour compared to the control group (p < 0.05). In the evaluation of apoptosis in semen samples, there was no significant difference between the control, 0.1 mM ferulic acid, and 10 mM L-glutamine groups (p > 0.05). As a result, it was determined that the addition of 10 mM L-glutamine to the spermatozoa extender increased the motility, viable spermatozoa, functional membrane integrity, intact acrosome ratios, or motility endurance after freeze-thawing and could be used successfully in the freezing extender of mouse spermatozoa.

Membrane remodulation and hyperactivation are impaired in frozen-thawed sperm of low-fertility bulls.

Bulls used in artificial insemination programmes worldwide undergo quality control checks, which are typically based on the evaluation of sperm motility and morphology. Despite this, some bulls can have lower than expected field fertility and the reasons for this remain to be elucidated. Here we hypothesised that sperm from bulls of varying fertility will differ in their ability to undergo capacitation-related events including an increase in membrane fluidity, protein tyrosine phosphorylation, hyperactivation and the acrosome reaction. Firstly, we used frozen-thawed semen from 10 high-fertility (HF) and 10 low-fertility (LF) bulls, and subjected them to in vitro capacitating conditions, following which sperm viability, membrane fluidity, acrosome integrity and protein tyrosine phosphorylation were assessed using flow cytometry. We then assessed the ability of sperm to undergo hyperactivation (induced using caffeine) utilising computer-assisted sperm analysis, and the acrosome reaction (induced using calcium ionophore) using flow cytometry. When sperm were incubated in capacitating conditions, a higher percentage of viable sperm from HF bulls exhibited high membrane fluidity when compared to LF bulls (8.8 ± 0.8% and 5.8 ± 1.2%, respectively; mean ± standard error; P < 0.05). There was no difference between fertility groups in the percentage of acrosome-reacted sperm following the incubation in in vitro capacitating conditions or following the induction of the acrosome reaction using calcium ionophore. However, more sperm from HF bulls became hyperactive in response to caffeine stimulation than sperm from LF bulls (21.6 ± 2.5% versus 14.1 ± 2.4%, respectively; mean ± standard error; P < 0.05). Taken together, sperm from LF bulls had an impaired ability to undergo membrane remodulation and to hyperactivate when induced in vitro. These are key events in the journey of sperm along the female reproductive tract and in the interaction with the oocyte and thus could explain the lower field fertility exhibited by some bulls.

Bacterial DNase activity as a putative inductor of sperm DNA fragmentation in infected bull frozen-thawed semen samples.

The aim of this study was to investigate the relationship between DNase activity associated with bacterial contamination of incubated bovine frozen-thawed spermatozoa and elevated sperm DNA fragmentation. Electrophoresis analysis of plasmid PBR322 incubated for 30 min at 37 °C with the supernatant of the diluent of frozen-thawed centrifuged bovine semen straws infected with bacteria showed clear evidence of DNase activity when compared to plasmid incubated in similarly prepared non-infected bovine diluent supernatant (Experiment 1). This DNase activity was subsequently found to be time dependent (0-60 min) and its activity prevented in the presence of EDTA (10 and 20 mM; Experiment 2). Semen straws infected (n = 10) and not infected (n = 10) with bacteria where incubated at 37 °C for up to 48h post-thaw. Semen infected with bacteria showed an exponential increase in bacterial growth and a corresponding increase in sperm DNA fragmentation. Non-infected semen samples showed no change in the incidence of sperm DNA fragmentation over the same period of incubation (Experiment 3). Our experiments reinforce the idea that exogenous DNases present in the semen should be considered as one of the primary contributing causes of sperm DNA fragmentation post ejaculation. In the case of the bull, post-thaw incubation of commercial straws contaminated with bacteria, resulted in increased levels of sperm DNA fragmentation, most likely associated with DNase activity (potentially restriction endonucleases) derived from the bacteria. Such adverse changes in sperm DNA fragmentation, as described here in vitro, may be also operative after insemination in the female reproductive tract (in vivo) and highlight the importance of implementing high levels of hygiene practice during semen processing, especially in light of future trends of bacterial resistance to the common antibiotics used in semen diluents.

Slow Cooling is Beneficial for Storage of Frozen-Thawed Equine Spermatozoa.

Cooled storage of semen after thawing can expand the use of frozen semen, providing the possibility of thawing and evaluating the semen at the storage site and subsequently shipping the semen. Our objectives were (1) to examine the motility and viability of frozen-thawed semen after cooled storage and (2) to compare two cooled-storage protocols for frozen-thawed semen. The samples (n = 31) were either placed immediately in a passive cooling box for 8 or 24 hours (CB) or placed in a refrigerator at 4°C for 30 minutes and then transferred to a passive cooling box (REF). Total and progressive motility were similar at T0 and T8-REF and at T0.5 and T8.5-REF. However, a significant reduction was observed in total motility (-8.12%) between T0 and T8-CB, and in total (-9.96%) and progressive motility (-8.52%) between T0.5 and T8.5-CB (P< .05). A significant reduction was also observed in total and progressive motility between T0 and T24, and between T0.5 and T24.5 for both storage protocols (CB and REF). Viability was lower in T8.5-CB (-11.87%), in T8.5-REF (-9.65%), in T24.5-CB (-13.52%), and in T24.5-REF (-12.32%) compared to T0.5 (P< .05). Our results demonstrate that sperm motility and viability decrease during cooled storage. However, storing the samples at 4°C for 30 minutes before placing the semen in a passive cooling box could mitigate the adverse effect of cooling during short-term storage (8 hours). Additionally, we observed individual variation between samples indicating that this protocol might not be suitable for all stallions. Our data shows that slow cooling and storage of frozen-thawed semen is a valid alternative that allows the expansion of frozen semen in breeding programs.

First study of in vitro protective effect of Lepidium meyenii (Maca) on frozen-thawed bovine spermatozoa.

Background and Aim: Lepidium meyenii Walp (Maca) is an herbaceous plant that grows in the Peruvian Andes and it has been widely used as a nutritional supplement and fertility enhancer and has been used in the treatment of a variety of diseases, such as rheumatism, respiratory disorders, and anemia. The most notable feature of Maca is its potent antioxidant capacity, which helps in the scavenging of free radicals and protection of cells from oxidative stress. This study aimed to evaluate the in vitro effect of Maca extract on thawed sperm cells from bulls. Materials and Methods: Three dilutions of 1, 10, and 100 mg/mL of Maca extract were incubated with frozen-thawed bovine semen and analyzed at 1, 3, and 24 h of exposure time, evaluating the activity of the extract on the DNA, motility, morphology, viability, integrity of the membrane and acrosome of spermatozoa. Results: The Maca extract improved the studied sperm parameters of motility, acrosome integrity, vitality, and DNA integrity of sperm cells at a concentration of 10 mg/mL, and at 1 mg/mL, an improvement was observed in the morphology and integrity of the membrane. However, the best activity of the Maca extract was observed on the DNA integrity of the sperm, which was effective at the three concentrations evaluated after 24 h of incubation. Conclusion: The results indicate that L. meyenii can help in maintaining spermatozoa cellular integrity after the frozen-thaw process, especially in the protection against DNA fragmentation. Therefore, Maca would be a feasible supplementation to protect sperm to maintain their fertile ability after thawing.

Topical application of cervix with hyaluronan improves fertility in goats inseminated with frozen-thawed semen.

OBJECTIVE: Artificial insemination plays an important role in genetic improvement in the goat farming system. The aim of this study was to investigate the effect of cervical application of hyaluronan (HA) on the fertility in goats after cervical artificial insemination using frozenthawed (F-T) semen. METHODS: After oestrous synchronisation with progesterone sponges and pregnant mare serum gonadotropin injection, both nulli- and multi-parous goats, were randomly allocated to 2 groups, and were inseminated with 0.25 mL of F-T semen (150×106 spermatozoa) twice at 52 h and 56 h after sponge removal. Prior to the insemination, goats in Group 1 only were given topical cervical HA application at 48 h after sponge removal. Site of insemination was recorded as os-cervix or intra-cervix or intra-uterus. Pregnancy was tested ultrasonographically 42 days after insemination. The data on pregnancy rates and percentage of animals according to the site of semen deposition were compared by Chi-square analysis. RESULTS: The overall pregnancy rate was significantly (p<0.004) higher in goats with prior application to the cervix with HA (63.3%) than without (36.0%). Same pattern was observed in the pregnancy rates of nulli- and multi-parous goats in both the groups. Percentage of nulliparous goats according to the site of insemination in the HA group did not differ between first and the second insemination. However, in multiparous goats the percentage of animals inseminated intra-cervically was significantly increased (p≤0.05) between the first and the second inseminations. CONCLUSION: The results suggest that significantly higher fertility rate in the "HA goats" compared to the "without HA" group was because of deeper insemination facilitated by topical cervical application of HA. The deeper insemination into the cervical canal increase the rate of fertilisation when the cervical artificial insemination is performed.

The Design of a New Catheter for Transcervical Artificial Insemination in Ewes.

In ovine species, transcervical artificial insemination (TCAI) is limited by the poor quality of frozen-thawed semen and by the convoluted cervical lumen hampering the passage of inseminating devices. The aim of the study was to test the efficiency of three newly designed catheters with bent tips of 3.5 mm, 5.0 mm or 8.0 mm in terms of reproductive performances (experiment 1) and to compare the results of TCAI with the best performing catheter of experiment 1 to those obtained in ewes submitted to surgical incision of cervical folds (SICF) prior to insemination (experiment 2). The following parameters were assessed: time to pass the cervix; depth of cervical penetration; site of deposition of semen; pregnancy (PR); and lambing rates (LR). The results of experiment 1 indicated that the 5.0 mm tip catheter resulted in deeper and faster TCAI and higher PR and LR compared to 3.5 mm and 8.0 mm tip catheters (p < 0.05). In experiment 2, TCAI with the 5.0 mm catheter did not differ from TCAI after SICF in terms of depth of semen deposition, time to pass the cervix, PR and LR (p < 0.05). In conclusion, the use of a catheter that allowed transcervical uterine deposition of semen without excessive manipulation led to satisfactory pregnancy rates.

Estrus synchronization and artificial insemination in Korean black goat (Capra hircus coreanae) using frozen-thawed semen.

Presently, there is an increased demand for livestock products all over the world which has led to more devotion on improving livestock population. Although goats have been bred for a long time in Korea, but there is not much research conducted on traditional Korean black goat (Capra hircus coreanae) compared to other livestock populations. Mutton consumption has been dramatically changing from medicinal use to edible meat and this trend directs the black goat populations declining and also mutton import quantities are increasing consistently. The present study introduced a new estrus synchronizing technique with subsequent artificial insemination (AI) for Korean black goats to enable crossbreeding with non-native breeds for the small or subsistent farmers. Our data highlighted that, the percentage of motile sperm from the electro-ejaculated samples declined significantly after freezing and melting. In addition, the sperm motility significantly declined with regard to sperm incubation period (0, 5, 60, and 120 min at 37°C) and was negatively correlated (64.2 ± 7.9%, 63.3 ± 5.8%, 49.9 ± 6.3%, and 35.9 ± 7.6%, respectively) in frozen-thawed sperm samples. Moreover, the E2 levels were unchanged even 24 h after controlled internal drug releas (CIDR) withdrawal. But, 48 h and 72 h after CIDR removal, E2 levels increased significantly. These data helps us to consider the two time points for AI; CIDR removal after 24 h, at which E2 decreases, and after 48 h, as the time at which progesterone increases. Additionally, the AI after 48 h of CIDR removal group exhibited significantly higher pregnancy and parturition rates (42.9%) compared to AI after 24 h after CIDR removal 28.6% group. In conclusion, these studies will propose an optimal estrus synchronisation process with subsequent timing of AI and also will promote the Korean black goat breeding industry.

Cryopreservation of Andalusian donkey (Equus asinus) spermatozoa: Use of alternative energy sources in the freezing extender affects post-thaw sperm motility patterns but not DNA stability.

The aim of this study was to compare the effect of three sugars and Equex paste in a freezing extender for donkey sperm cryopreservation. Ejaculates (n = 18) were collected from six Andalusian donkeys of proven fertility were pooled (two ejaculates per pool) and cryopreserved using a freezing extender containing three different sugars (glucose, fructose and sorbitol), with or without the addition of Equex paste. Sperm quality was assessed before and after freezing-thawing for motility, morphology, plasma membrane integrity, acrosome integrity and DNA integrity. The use of sorbitol in the freezing extender improved total and progressive sperm motility (P < 0.05) and amplitude of lateral head displacement (P < 0.01), but it reduced the values for other sperm motility variables compared with glucose (P < 0.001). The use of fructose resulted in a reduction in values for most CASA variables (P < 0.05), whereas addition of Equex paste did not have any beneficial effect on values for these variables (P > 0.05). Glucose was more effective in maintaining sperm morphology (P < 0.05), while there was no beneficial effect with the addition of Equex paste (P > 0.05). Supplementation of fructose and Equex paste in the freezing extender decreased plasma membrane integrity (P < 0.05) as compared with glucose, but there were no differences between treatments for acrosome and DNA integrity (P > 0.05), even after 24 h of incubation. The use of different sugar sources in the extender could affect the in vitro post-thaw quality of cryopreserved donkey spermatozoa, with sorbitol being an interesting alternative for improving the sperm quality. Results of the present study indicate the use of Equex paste could negatively affect post-thaw outcomes for sperm viability in this species.

Surgery on cervical folds for transcervical intrauterine artificial insemination with frozen-thawed semen enhances pregnancy rates in the sheep.

In sheep industry, genetic progress rate achieved by artificial insemination (AI) is limited by the convoluted anatomy of the cervix, which does not allow the passage of an insemination catheter for uterine semen deposition. The aim of this study was to test, in 98 pregnant at term Sarda ewes, the effects of: Experiment 1) total or partial ablation of cervical folds and Experiment 2) 4 or 2 incisions of cervical folds, on the passage of an insemination catheter, deposition of frozen-thawed semen and pregnancy rates. Surgical procedures were performed within 24 h from parturition providing deep sedation and epidural anaesthesia. Duration of surgeries and post-operatory recovery were carefully monitored. For both experiments, 5 months since surgery, independently of the stage of oestrus cycle, cervical patency was tested through the transcervical passage of a palpation probe. Six months since surgery, in Experiment 1, ewes were naturally mated with fertile rams. In Experiment 2, ewes submitted to incisions of the cervical folds and a control group underwent synchronisation of oestrus and transcervical AI with frozen-thawed semen. Thirty days later, for both experiments, pregnancy rates were assessed by ultrasonography and lambing rates were recorded. Five months after surgery, in Experiment 1, transcervical passage of a palpation probe to reach the uterine lumen was possible in all ewes submitted to total and partial ablation of folds. In Experiment 2, this was achievable in 90.5% ewes with 4 incisions of the folds and in 89.6% ewes with 2 incisions with no significant differences among groups (P = 0.44). In Experiment 1, pregnancy rates in ewes mated to rams after total or partial ablation of the cervical folds was 100%. In Experiment 2, following transcervical AI, pregnancy rates were higher in groups submitted to 4 (63.7%) or 2 (41.4%) incisions of the cervical folds compared to the control group (8%; P<0.05). These data were confirmed at lambing with rates of 56.8% and 41.4% in ewes submitted to 4 or 2 incisions respectively, significantly higher than the control group (4%; P<0.05). Surgical ablation or incision of the cervical folds in post-partum ewes represent valid procedures for transcervical intrauterine deposition of semen for AI, obtaining satisfactory pregnancy rates. These procedures might be useful in programs of genetic selection and MOET.

Genetic parameters and estimated breeding values for traits of raw and frozen-thawed semen in German Warmblood stallions.

Objectives of the present study were to estimate genetic parameters for frozen-thawed semen traits of 271 fertile German Warmblood stallions and genetic correlations with raw semen quality traits. Semen samples were collected from stallions utilized for semen collection and artificial insemination (AI) on the Lower Saxon National Stud Celle and the North Rhine-Westphalian National Stud Warendorf. Semen quality variables were analyzed in 63,972 raw (gel-free volume, concentration, progressive motility, number of sperm) and 3681 frozen-thawed samples (motility, DNA fragmentation index (DFI), non-viable sperm). A multivariate linear animal model was used to estimate additive genetic and permanent environmental variances among stallions as well as estimated breeding values (EBVs) for all semen traits. Heritability estimates were greatest for DFI (h2 = 0.45) and least for non-viable sperm counts ( h2 = 0.11). Additive genetic correlations between progressive sperm motility in raw semen and DFI (rg = -0.79) as well as non-viable sperm (rg = -0.45) were negative. The EBVs for frozen-thawed semen traits ranged from 49 to 181 with mean reliabilities of 0.28 to 0.43. The EBVs for progressively motile sperm post-thawing and DFI were the most highly correlated traits with EBVs for stallion fertility (r = 0.38 and r = -0.17). Stallions with relatively greater EBVs for progressive motility in raw semen may be most suitable when freezing semen for storage and subsequently thawing it for AI. Using EBVs for semen traits in selection of stallions to AI mares appears as an option for genetic improvement to enhance fertility after AI.

Effect of increasing equilibration time of diluted bull semen up to 72 h prior to freezing on sperm quality parameters and calving rate following artificial insemination.

An equilibration period of approximately 3-4 h prior to semen cryopreservation is standard practice for maintaining membrane integrity and motility of bull sperm. However, a number of studies indicate that an overnight equilibration period prior to freezing results in improved post-thaw semen quality thus optimising pregnancy rates. The aim of this study was to assess the effect of increasing the equilibration time of bull semen up to 72 h before freezing on sperm quality parameters and calving rate (CR) following artificial insemination (AI) with frozen-thawed semen. The effect of holding semen at 4 °C for 6, 24, 48 or 72 h post dilution before freezing on subsequent post-thaw total and progressive motility (Experiment 1) and field fertility (n = 1640 inseminations, Experiment 2) of frozen-thawed semen was assessed. Equilibration time did not affect post-thaw total and progressive motility (P > 0.05). In addition, there was no effect (P > 0.05) of equilibration time on field fertility with a CR of 53.3, 50.5, 51.3 and 47.3 for the 6, 24, 48 and 72 h treatments, respectively. In conclusion, increasing the equilibration time of diluted bull semen from 6 to 72 h had no significant effect on CR, within the expected range of fertility outcomes, thus providing semen processing centres with flexibility in the time which semen can be held prior to freezing.

Combined effect of apigenin and ferulic acid on frozen-thawed boar sperm quality.

The main aim of the present study was to evaluate the cryoprotective effect of apigenin (AP) and ferulic acid (FA) on boar sperm during cryopreservation. AP and FA were both demonstrated to be high-efficiency antioxidants and had not previously been used to protect sperm from cryodamage. As boar sperm is sensitive to oxidative stress, suitable antioxidants are still needed for improving frozen-thawed sperm quality. With this purpose, semen samples coming from five boars were used in this study. Ejaculates of five boars were mixed and split into 16 aliquots, in which different doses of AP and FA were added separately or together. The motility, the plasma membrane integrity, the mitochondrial activity, the acrosomal integrity, the antioxidase activities and the malondialdehyde concentration of the frozen-thawed boar sperm were assessed. The results suggested that both AP and FA significantly improved the frozen-thawed boar sperm quality in all these aspects when they were added to the freezing extender separately, while the highest improvement was recorded when the extender was supplemented with 0.1 mmol/L AP plus 0.15 mmol/L FA. These findings demonstrated that supplementation of freezing extender with both AP and FA had a combined, beneficial effect on frozen-thawed boar sperm.

Cryoprotective effect of glutamine, taurine, and proline on post-thaw semen quality and DNA integrity of donkey spermatozoa.

This study was conducted to evaluate the effect of amino acid addition to semen on post-thaw quality of donkey spermatozoa. Eighteen ejaculates were pooled and divided into aliquots which were cryopreserved in Gent A® containing 1% ethylene glycol (Gent-EG) and supplemented with 0 (as control), 20, 40, or 60 mM of glutamine, proline, or taurine. The greatest concentration (60 mM) of glutamine and taurine resulted in greater (P < 0.001) post-thaw sperm motility. Amino acid supplementation did not improve (P > 0.05) sperm morphology and membrane plasma integrity compared with the control samples. Whereas, improvement (P < 0.05) of acrosome integrity was observed with use of 60 mM glutamine. After thawing, there were no differences (P > 0.05) in the sperm DNA fragmentation index (sDFI) among treatments. The 60 mM glutamine and 40 mM taurine treatments, however, resulted in a reduction (P < 0.05) in sDFI values in the first 6 h of semen incubation, compared with the control samples. At 24 h, the sDFI values were less (P < 0.05) in all supplemented as compared with control samples, except for the 20 mM proline treatment group. In conclusion, supplementation of the Gent-EG extender with glutamine or taurine at 60 mM improved post-thaw donkey sperm quality. The addition of proline to the freezing extender, however, did not provide any significant enhancement in sperm quality, compared with the control group.

Comparison of plant- and egg yolk-based semen diluents on in vitro sperm kinematics and in vivo fertility of frozen-thawed bull semen.

Diluents using components of plant origin have been developed as an alternative to animal based extenders for the dilution of bull semen, however, it is unclear if use of these diluents results in in vivo fertility rates similar to those that occur with use of traditional egg yolk-based diluents. The aim of this study was to assess the effect of semen diluent on 60-day non-return rate (NRR) following artificial insemination (AI) with frozen-thawed bull semen. The effect of semen dilution in one of three different commercial diluents (BullXcell - egg yolk-based, OptiXcell - plant-based or AndroMed - plant-based) on post-thaw total and progressive motility as well as kinematic parameters (Experiment 1) and field fertility (Experiment 2, n = 1,480 inseminations) was assessed. Semen stored in OptiXcell had greater post-thaw total and progressive motility than AndroMed (P < 0.05) but did not differ from BullXcell. Semen stored in BullXcell had a greater beat cross frequency and straight line velocity compared to semen stored in AndroMed (P < 0.05) but did not differ when compared with use of OptiXcell; while values for these variables when using OptiXcell and AndroMed did not differ from each other (P > 0.05). There was no difference in any other sperm kinematic parameters (P > 0.05). There was no effect of diluent on 60-day NRR (71.5%, 67.8% and 70.6% for BullXcell, OptiXcell and AndroMed, respectively). In conclusion, while diluent significantly affected post-thaw sperm motility and kinematics, no effect on 60-day NRR was observed. Given that OptiXcell and AndroMed are animal protein-free media these diluents may be a suitable alternative to BullXcell for the storage of frozen-thawed bull semen.