Horse Semen Research Articles

The optimum concentration of soya-lecithin for replacing egg yolk in INRA-82-modified semen extenders improved the cryopreserved semen quality and DNA integrity of Arabian stallions

The cryopreservation of stallion spermatozoa varies greatly between animals. Different equine semen extender supplements were invented to maximize the post-thaw semen quality. Soy lecithin is one of those additives that contain plant origin phospholipids used to replace egg yolk to minimize disease transmission. This study aimed to determine which concentrations of soy lecithin can replace egg-yolk in modified INRA-82 semen extenders for the cryopreservation of horse semen. Semen was collected from six Arabian stallions and extended with modified INRA-82 semen extender supplemented with 0.0, 1.25, 2.50, and 5.0% soya lecithin. Frozen-thawed sperm motility was measured at 0, 1, 2, and 3 h. Viability index, sperm plasma membrane integrity (SPMI), and acrosome integrity in addition to the comet assay non-fragmented DNA, head DNA, tail DNA, tail length, and tail moment were statistically analyzed. The addition of 2.5% soya lecithin had the highest post-thaw sperm motility at 0, 1, 2 h (P < 0.001) and 3 h (P < 0.0001), the optimum viability index (P < 0.0001), SPMI (P < 0.05), acrosome integrity (P < 0.0001), non-fragmented DNA (P < 0.01), DNA in the comet head (P < 0.05), minimum DNA in the comet tail (P < 0.05), shortest comet tail length (P < 0.0001), and lowest comet tail moment (P < 0.01). Soy lecithin at concentrations other than 2.5% deteriorated all post-thaw semen parameters. Soy lecithin (2.5%) can replace egg yolk in the modified INRA-82 semen extender for the cryopreservation of good-quality stallion semen.

Shipping duration and temperature influence the characteristics of cryopreserved horse semen stored in different shipping devices for up to 14 days

This study aimed to investigate the effects of storing horse semen either in a dry shipper (≤ −150 °C) or on dry ice (≤ −78 °C) for up to 14 days. A total of 264 frozen semen straws from male horses (n = 8) stored in liquid nitrogen were transferred on day 0 (d0) to a dry shipper or a dry ice styrofoam box. On d1, d3, d7, d10, and d14, straws from the dry shipper and dry ice were returned to the liquid nitrogen container. Semen was evaluated by CASA for total (TMot), progressive motility (PMot) and sperm velocity parameters, by fluorescence microscopy for percentage of membrane-intact sperm (SYBR14/PI), high mitochondrial membrane potential (HMMP; JC1) and DNA fragmentation. Temperature inside the containers was monitored continuously. Until d7, no changes were observed in TMot, PMot, and membrane-intact spermatozoa. Thereafter, all three parameters decreased in semen stored on dry ice but not in a dry shipper (time p < 0.001, time x shipping device p < 0.001). The HMMP decreased continuously over time in both containers with a more pronounced decrease on dry ice compared to the dry shipper (shipping device p < 0.01, time p < 0.001, time x device p < 0.001). The DNA fragmentation increased on d10–14 on dry ice and d14 in the dry shipper (time p < 0.001, time x device p < 0.01). In conclusion, frozen horse semen can be safely stored for up to 7 days on dry ice. Sperm DNA integrity and HMMP, however, were adversely affected after 14 days in both shipping devices.

The lipidomic profiles of donkey semen present greater diversity and abundance than horse semen

While donkeys and horses belong to the same family and are able to cross-mate to produce hybrids, there are many unique features of either species regarding their sexual behavior, semen characteristics, and tolerance to cryopreservation; therefore, a better understanding of the physiology of either species can enhance semen processing and fertility. Lipids play a paramount role in the fluidity and flexibility of the sperm plasma membrane and are intrinsically related to sperm motility and plasma membrane integrity. This study aimed to determine the lipid profile of donkey and horse semen. Semen from 19 jacks and 25 stallions was collected by artificial vagina, and gel-free semen was assessed for motility, morphology, concentration, and volume. An aliquot of gel-free semen (50 million sperm/mL) was placed in cryovials and then plunged into liquid nitrogen until lipidomic analysis. The lipidomic profile of the gel-free semen was performed with MALDI-MS with no addition of an extender. The analyses were performed with MetaboAnalyst 2.0 and the discriminating analyses of PLS-DA to demonstrate differences between horses and donkeys. The lipid types that were differentially expressed were analyzed by ANOVA followed by Tukey test. The Volcano plot was used to compare species, using the criteria values of P<0.05 andfold-change (FC) ≥1.5. A total of 101 ion lipids were found in horse semen and 105 in donkey semen. Five ion-lipids with mass charge (mz) 762, 822, 865, 867, and 879 were uniquely present in horse semen, but not identified in the databases (www.lipidmaps.org), whereas 9-ion-lipid were present in donkey semen (m/z 709.4, 731, 753, 756, 778, 796, 798, 811 and 880) with only 709.4 being identified (Phosphatidylglycerol). The donkeys showed 18 ion-lipid with greater relative abundance (P<0.05 and fold-change>1.5), 12 identified in the database (4 triacylglycerols, 2 phosphatidylglycerols, 2 phosphatidylcholine, 2 phosphatidylinositols and 2 sphingolipids); whereas the horse had ten ion-lipid with greater abundance (P<0.05 and fold-change >1.5) but only 4 identified (2 diacylglycerols, 1 triacylglycerol and 1 phosphatidylglycerol). Among these identified lipids, 10 have one or more double bonds, making them mono- or polyunsaturated, while in horses only 1 was polyunsaturated. This may be one of the underlying reasons for lower tolerance to cryopreservation/cooling of donkey semen. In conclusion, the lipidomic profile of donkey and stallion semen differ with regard to relative abundance and diversity.

Uterine inflammatory response, embryo recovery and morphometry in interspecies breeding

Problem mares presumably have better fertility when bred with donkeys; this could be due to differential post-breeding uterine inflammatory response modulated by seminal plasma (SP) or embryonic ultrastructural features; however, these hypotheses have not been studied. This study aimed to assess the: (i) uterine inflammatory response; (ii) embryo recovery and morphometry; (iii) the potential crosstalk between the proteomic and metabolomic of SP and uterus of mares bred with donkey or horse semen or infused with the SP of either species. Twenty-two mares were randomly assigned in a crossover design, with a washout cycle in-between, to receive semen and SP from both species and saline in five different estruses. The uterine inflammatory response was assessed via fluid accumulation measured by ultrasound, neutrophils on uterine cytology, and cytokines on uterine fluid (IL-1a, IL-1b, IL-4, IL-6, CXCL-8, IL-10) at 0-, 6, 24, 48-h-post-AI/infusion. Embryos were recovered 8d post-ovulation, classified, graded, measured, and the nuclei morphometrics were assessed with ImageJ on images acquired with a fluorescent microscope. Proteomics and metabolomics of SP were identified with LC-MS/MS and analyzed with R-studio, PANTHER, and Perseus. A mixed linear model and Kruskal-Wallis rank-sum test was used for normally and not-normally distributed variables. Uterine fluid increased at 6-h post-infusion/AI (P<0.05) but did not vary across groups (P>0.05). There was an effect of time (P=0.001) and of time*groupinteraction (P<0.001) but no effect of group (P=0.86) on neutrophil counts. At 6h post-infusion, the neutrophil count in the mares infused with donkey-SP tended to be the lowest (P=0.07). Cytokine concentrations peaked at 6h (P<0.05); horse semen triggered higher IL-6 and lower IL-1b concentrations than donkey semen (P<0.05). Donkey- and horse-SP shared 16 proteins; each species had 99 and 64 unique proteins, respectively. The metabolomic fingerprinting revealed a higher number of anti-inflammatory metabolites, such as choline, epinephrine, palmitoleic acid, and palmitic acid, in donkey-SP compared to horse-SP, suggesting these molecules can play a role in the uterine inflammatory response. Embryo recovery was similar between donkey-bred (59%) and horse-bred (55%) (P<0.05). Horse and mule embryos had similar sizes, grades, number of compact nuclei per sector, fragmented nuclei index mule, and micronuclei index (P>0.05); however, the mitotic nuclei index was higher in mule embryos (P=0.03). In conclusion, this is the first study to compare the uterine inflammatory response, embryo recovery, and morphology of mares bred with donkeys and horses. Horse semen triggered a greater uterine inflammatory response than donkey semen, and donkey-SP tended to have the lowest. The SP composition may explain the post-breeding uterine inflammatory response. Equid embryos share similar nuclear ultrastructure features, except that mules have a higher mitotic index.

Correlation of equine sperm kinetic parameters with fertility index

In horses, fertility has a great economic impact on reproductive management and several studies have been carried out in order to optimize the success rate of reproductive biotechnologies. For stallions, reliable prediction of fertility by semen evaluation is therefore required, such as kinetics, membrane integrity and morphology. Analysis of total sperm motility is one of the most important characteristics assessed in horse semen worldwide. With the aim to reduce the subjectivity of this method, computerized seminal analysis systems (CASA) was introduced. Even with more sophisticated motility assessments, however, CASA cannot accurately predict semen sample fertility (Amann et al., 2014). The objective of this work was to retrospectively analyze the correlation between kinetic parameters such as total motility (TM), progressive motility (PM) and rapid spermatozoa (RAP) of stallions and their fertility rates. We analyzed 22 publications, theses and dissertations from the Center for Biotechnology and Animal Reproduction (CERAN)- UNESP, Botucatu, São Paulo, published between 2008 and 2017. The works provided data on kinetic parameters, MT, MP and RAP, (VAP >70 μm/s and STR >80%) by computerized analysis CASA (Hamilton Thorn Research – IVOS ® 12, Beverly, Massachusetts, USA) of refrigerated and frozen semen from approximately 44 stallions,together with information on their in vivo fertility by insemination of mares (N=372) under different managements conditions. For statistical analysis, Spearman's determination test and correlation coefficient were used (software GraphPad). In average, 3.4±3.2 stallions were used with 16.9±6.1 mares per publication. MT was 60.2±17.9%, MP 26.9±11.5%, and RAP 48.2±16.7%, respectively. The average fertility in the studies was 52.1±25.9%. Fertility rate was determined per cycle, with ultrasound performed 15 days after insemination. A strong correlation was found for fast spermatozoa with fertility rate (r = 0.74; p<0.0001) and moderate correlations for PM and TM (r = 0.61 and 0.53; p<0.01, respectively) as well as TM, PM and RAP, (R2= 0.37; R2=0.42; R2= 0.46; respectively) and p<0.01. This retrospective study demonstrates that the three parameters TM, PM and RAP are positively correlated and RAP had the greatest association with fertility. In conclusion, among the analyzed variables, RAP is most consistent with the fertility of stallion semen. Acknowledgements: The authors are grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – (CAPES).

Applicability of 2,4-dinitrophenylhydrazine (DNPH) method of protein oxidative damage measurement in the seminal plasma of canine ( Canis lupus familiaris) and stallion ( Equus caballus).

Seminal plasma (SP) proteins are responsible for sperm functional quality. Developing a reliable method to determine the degree of oxidative damage of these proteins is important for establishing semen fertilizing ability. The main aim of the study was to verify the applicability of protein carbonyl derivatives measurement in the SP of canine and stallion, using a method with 2,4-dinitrophenylhydrazine (DNPH). The research material consisted of ejaculates obtained from eight English Springer Spaniels, and from seven half-blood stallions during the breeding and non-breeding season. The content of carbonyl groups in the SP was measured on the basis of the reactions with DNPH. The following reagent variants were used to dissolve protein precipitates: Variant 1 (V1) - 6M Guanidine solution and Variant 2 (V2) - 0.1M NaOH solution. It has been shown that to obtain reliable results for the measurement of protein carbonylated groups in the dog and horse SP, both 6M Guanidine and 0.1M NaOH may be used. A correlation was also found between the number of carbonyl groups and the total protein content in the canine (V1: r = -0.724; V2: r = -0.847) and stallion (V1: r = -0.336; V2: r = -0.334) SP. Additionally, the study showed a higher content (p≤0.05) of protein carbonyl groups in the stallion SP in the non-breeding season compared to the breeding season. The method based on the reaction with DNPH, due to its simplicity and cost effectiveness, appears to be suitable for large-scale application in the determination of the SP proteins oxidative damage in dog and horse semen.

ProAKAP4 Concentration Is Related to Sperm Motility and Motile Sperm Subpopulations in Frozen-Thawed Horse Semen.

ProAKAP4 is the precursor of AKAP4 (A-kinase Anchor protein 4), the main structural protein of the fibrous sheath of sperm. The amount of proAKAP4 reflects the ability of spermatozoa to maintain the flagellum activity and functionality up to the site of fertilization and is positively correlated with progressive motility in several mammalian species. The aim of this study was to investigate the relationship between proAKAP4 concentration with horse sperm motility descriptors and spermatic motile subpopulations. For this purpose, a total of 48 ejaculates from 13 different stallions were analyzed. Spermatic motility descriptors were obtained by the CASA system, and four motile subpopulations (SP) with specific motility patterns were statistically identified. ProAKAP4 concentrations were evaluated by ELISA. The relationship between motility descriptors of sperm subpopulations and proAKAP4 concentrations was evaluated. Following a hierarchical cluster statistical analysis, ejaculates were divided into two groups according to their proAKAP4 concentrations, either having low proAKAP4 concentrations (5.06−35.61 ng/10M spz; n = 23) or high (39.92−82.23 ng/10M spz; n = 25) proAKAP4 concentrations (p < 0.001). ProAKAP4 concentrations were positively correlated (p < 0.05) with total and progressive motility, as well as with parameters of velocity. ProAKAP4 amount also showed a negative correlation (p < 0.05) with sperm motile subpopulation number 3, which was the subpopulation with the lowest velocity parameters. In conclusion, proAKAP4 concentration in stallion semen positively reflects sperm progressive motility with the functional velocity kinematic descriptors. Concentrations of proAKAP4 higher than 37.77 ng/10M spz were correlated with a very good quality frozen/thawed stallion semen.

The interference of ozone gas in kinects and mitochondrial potential of equine sperm submitted on cryopreservation.

The objective of this study was to evaluate the effects of the addition of different concentrations of ozone to quarter horse semen submitted to cryopreservation. Six ejaculates from four stallions were collected and were divided in four experimental groups: a control group (BotuCRIO® extender) and three other groups with BotuCRIO® ozonized at concentrations of 6, 8 and 12 μg of O3/mL. The semen samples were diluted (200 x 106 spermatozoa/mL), filled in straws and frozen. After thawing (37 ºC, 30s), the samples were evaluated at 0, 30 and 60 minutes of incubation regarding sperm kinetics by a computer-assisted sperm analysis (CASA), and plasma membrane integrity (PMI), acrosome integrity (ACi) and mitochondrial membrane potential (MMP) by fluorescent probes. There was a reduction in the kinetic parameters total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) in all groups during the thermoresistance test (TT), a pattern also found in PMI and MMP analyses (p<0.05). There was no difference (p>0.05) between the control and treatment (6, 8, and 12 μg of O3/mL) groups, in any of the evaluated times for the kinetic parameters TM, linearity (LIN), straightness (STR), wobble index (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). Regarding the VCL, VSL and VAP parameters, the group treated with 6 μg did not differ from the control or from 8 μg, but was higher than 12 μg at 30 and 60 minutes. ACi and PMI did not differ between groups (p>0.05), but PMI was lower in groups 8 μg and 12 μg compared to the control and 6 μg (p<0.05). It was concluded that the addition of ozone does not present beneficial effects for cryopreservation of equine semen at the concentrations used and decreases important parameters of fertility.

Evaluation of Low-density lipoprotein and egg yolk as cryoprotectants non-penetrating of Peruvian Paso horse semen

Evaluation of Low-density lipoprotein and egg yolk as cryoprotectants non-penetrating of Peruvian Paso horse semen

Functional Assessment of Diluent Choice for Semen Cryopreservation from Stallions with High and Low Freezability

Background: fertility rates using horse frozen-thawed semen remain lower than in other livestock species. This fact further suggests that horse semen hold intrinsic sensitivity to cryoinjury that must be investigated. Moreover, there is a substantial influence of genetic factors and diluent choice upon horse cryopreservation outcome. Collectively, these genetic and technical properties of horse semen could be explored to identify factors or conditions that may increase semen viability after freeze-thawing. The aim of this work was to evaluate the effect of diluents Botu-Crio®,Lactose-EDTA®, and INRA-82® on cryopreserved semen from stallions with high (HFA) and low freezability (LFA).Materials, Methods &amp; Results: frozen-thawed semen was evaluated for motility, membrane integrity, and sperm DNA fragmentation using the thermoresistance test (TRT). Comparisons for each parameter were done in a pair-wise fashion between HFA and LFA semen at one-hour intervals during the TRT (0 h - 4 h). Sperm motility in HFA, regardless of the diluent, was larger (P &lt; 0.05) than LFA, both on 0h and 1h. In the 2h evaluation, sperm motility using Botu-Crio® and Lactose-EDTA® was greater (P &lt; 0.05) for HFA. Analysis of sperm membrane integrity was similar between HFA and LFA semen (P &gt; 0.05) at 0 h and 3 h. Sperm DNA fragmentation was lower (P &lt; 0.05) in HFA semen at 0 h and 1 h. Discussion: frozen-thawed semen from stallions of high freezing ability showed greater motility at all analysis, irrespectively of diluent choice, suggesting a strong influence of genetic factors on cryopreservation outcome. Membrane integrity was similar immediately after thawing but did differ later on other TRT time-points, irrespectively of diluent choice. As observed for motility, it was expected that sperm cells of stallions of HFA would show higher membrane integrity than their LFA counterparts. Sperm DNA fragmentation was quite low for both groups, as described in horses. Surprisingly, sperm DNA fragmentation incidence was constant throughout the analysis for both HFA and LFA. It was initially envisioned that increased DNA fragmentation would be found in semen from LFA stallions, since it is caused by multiple origins such as genetic factors. In conclusion, the semen diluent affects horse sperm motility after thawing, particularly from stallions with lower semen freezability.

Cryopreservation of horse semen with a liposome and trehalose added extender

The aim of the study was to evaluate the effect of cryopreserved equine semen in the presence of trehalose-loaded liposomes on the integrity and function of sperm cells. Six ejaculations of five stallions collected with an artificial vagina were used. The ejaculates were diluted with INRA 96® 2:1 v/v and transported at 22 °C to the laboratory. Before cryopreservation, the semen was diluted with INRA Freeze® to obtain the following treatments: T1) INRA Freeze® (control), T2) INRA Freeze® + liposomes, T3) INRA Freeze® + liposomes+trehalose. Data were analysed using the Kruskal Wallis test. The percentages of sperm with intact DNA were 54.5, 57.9, and 64.8% for T1, T2 and T3, respectively (P>0.05). When evaluating the acrosomal and capacitation state after filtering with Percoll®, the percentages of spermatozoa without acrosome reaction and without capacitation were 67.8, 79.2 and 68.1% in T1, T2, and T3, respectively (P>0.05), while the capacitated sperm without acrosome reaction and without capacitation was similar in T1 (47%) and T3 (32%) (P>0.05), and lower in T2 (16%) before filtering with Percoll®. The use of liposomes and liposome-trehalose did not affect on the functional status and nuclear chromatin of the equine sperm after freezing, but it did affect the percentage of capacitated sperm without acrosome reaction after selecting the thawed semen using the Percoll® gradient.

Effect of Gum Arabic on Stallion Sperm Survival During Cold Storage and Post Freezing

Abstract This study is aimed at investigating effects of supplementation of stallion’ semen extender with various concentrations of Gum Arabic (GA) versus egg yolk (EY) on viscosity, sperm motility and survival during cooling and freezing. Physical sperm characteristics; i.e. curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN) and straightness index (STR) were evaluated. Based on the sperm velocity (velocity of the average path), individual spermatozoons were classified into two major groups; i.e., progressively motile (&gt;45 μm/sec) and immotile (0-45 μm/sec) spermatozoa. Addition of 3, 9 or 15% of GA to HF-20 extender resulted in linear decreases in VCL, VSL and VAP and a decrease in the percentage of progressively motile spermatozoa. Dilution of horse semen samples with high viscosityextenders (i.e., high percentage of GA) decreased the VCL, VSL and VAP in fresh and chilled semen. Freezing semen in high viscosity-extenders reduced percentage of progressively motile spermatozoa compared with those of low viscosity-extenders. In refrigerated and frozen semen samples, the extender containing 15% GA had detrimental effects on the percentage of progressively motile sperm cells and velocity of progressive motile sperm. Moreover, cooling sperm in extenders containing 9 or 15% of GA for 72 hours resulted in complete motility cessation. In conclusion, GA could replace EY in stallion semen extenders at a level of 3% to maintain the physical and biological characteristics of cold and frozen semen.

Does MnTBAP ameliorate DNA fragmentation and in vivo fertility of frozen-thawed Arabian stallion sperm?

Overproduction of reactive oxygen species during sperm freeze-thawing process leads to membrane lipid peroxidation, DNA damage, motility loss, and subsequent death. This oxidative stress can be alleviated by the addition of some antioxidants to semen extenders prior to freezing. This study was performed to evaluate the in vitro effectiveness of MnTBAP (a cell permeable antioxidant) on stallion sperm freezability and in vivo fertility rate. Twenty-one ejaculates were, collected with missouri model artificial vagina (n = 3 stallions, seven ejaculate each), and diluted (1:2 v/v) with phosphocaseinate base INRA extender, containing 0 (control), 100, 200 and 300 μM of MnTBAP and frozen using acontrolled-rate freezing system. The following parameters were determined: sperm motility, viability, membrane integrity, acrosome abnormalities, lipid peroxidation and DNA fragmentation. MnTBAP improved horse semen quality parameters in a dose-dependent manner. The100 μM concentration of MnTBAP did not show a significant difference in semen parameters compare with control group (p > 0.05). Accordingly, the extender supplemented with 200 μM resulted in higher sperm total and progressive motility (55.3 ± 4.28% and 33.2 ± 2.90%), viability (43.9 ± 2.14%), and membrane integrity (50.8 ± 2.14%), provided a greater protective effect in the percentage of total abnormalities compare to other groups (p < 0.05), and showed lower sperm with damaged DNA with lower MDA levels (p < 0.001). Higher concentrations (300 μM) not only did not improve the results but inversely affected sperm parameters. Twelve mares were used for fertility trial in the cross over study of 60 deep horn inseminations performed using control (9/30 pregnancy/mare) and 200 μM - MnTBAP (14/30 pregnancy/mere) groups frozen semen. The Average pregnancy rates were not significantly different between control and treated group (30% and 46.66%respectively) (p > 0.05). Under the conditions of this study, 200 μM - MnTBAP reduced the oxidative stress and protect the DNA fragmentation of Arabian stallion sperm during cryopreservation; but did not improved pregnancy rates.

Effect of Extenders and Insemination Protocol on the Fertilizing Capacity of Cryopreserved Arabian Horse Semen

Effect of Extenders and Insemination Protocol on the Fertilizing Capacity of Cryopreserved Arabian Horse Semen

Cytosine methylation of sperm DNA in horse semen after cryopreservation

Semen processing may contribute to epigenetic changes in spermatozoa. We have therefore addressed changes in sperm DNA cytosine methylation induced by cryopreservation of stallion semen. The relative amount of 5-methylcytosine relative to the genomic cytosine content of sperm DNA was analyzed by ELISA. In experiment 1, raw semen (n = 6 stallions, one ejaculate each) was shock-frozen. Postthaw semen motility and membrane integrity were completely absent, whereas DNA methylation was similar in raw (0.4 ± 0.2%) and shock-frozen (0.3 ± 0.1%) semen (not significant). In experiment 2, three ejaculates per stallion (n = 6) were included. Semen quality and DNA methylation was assessed before addition of the freezing extender and after freezing-thawing with either Ghent (G) or BotuCrio (BC) extender. Semen motility, morphology, and membrane integrity were significantly reduced by cryopreservation but not influenced by the extender (e.g., total motility: G 69.5 ± 2.0, BC 68.4 ± 2.2%; P < 0.001 vs. centrifugation). Cryopreservation significantly (P < 0.01) increased the level of DNA methylation (before freezing 0.6 ± 0.1%, postthaw G 6.4 ± 3.7, BC 4.4 ± 1.5%; P < 0.01), but no differences between the freezing extenders were seen. The level of DNA methylation was not correlated to semen motility, morphology, or membrane integrity. The results demonstrate that semen processing for cryopreservation increases the DNA methylation level in stallion semen. We conclude that assessment of sperm DNA methylation allows for evaluation of an additional parameter characterizing semen quality. The lower fertility rates of mares after insemination with frozen-thawed semen may at least in part be explained by cytosine methylation of sperm-DNA induced by the cryopreservation procedure.

Substitution of egg yolk by a cyclodextrin-cholesterol complex allows a reduction of the glycerol concentration into the freezing medium of equine sperm

The aim of this work was to completely replace the egg yolk a classical diluent for freezing equine semen by a cyclodextrin-cholesterol complex. At the same time, the reduction in the glycerol content used for cryopreservation and the incubation time between sperm and the freezing media were evaluated.Horse ejaculates were frozen with four different freezing extenders: a frozen reference medium (IF) containing egg yolk and 2.5% glycerol and media without egg yolk but supplemented with 1.5 mg 2-hydroxypropyl-beta-cyclodextrin cholesterol (HPβCD-C) complex and containing either 1% (G1), 2% (G2) or 3% glycerol (G3). Three incubation times (90, 120 and 180 min) at 4 °C between the fresh semen and the different media were tested before freezing. Viability and motility analyses were performed with computer assisted semen analysis (CASA).Results showed that the freezing media containing the HPβCD–C complex with 1%, 2% and 3% glycerol significantly improve the 3 in vitro parameters of post thawing semen quality (viability, progressive and total mobilities) compared to IF. The best improvement of the parameters was obtained with G1 medium and the longest contact time. The substitution of egg yolk by HPβCD–C complex allows the decrease of protein charge of the medium while favouring the cholesterol supply to membrane spermatozoa offering it a better resistance to osmotic imbalance and a better tolerance to the glycerol toxicity. Our results highlight that the egg yolk of an extender for the freezing of horse semen can be completely substituted by HPβCD–C complex.

Characteristics and freezability of Kathiawari horse semen

Semen was collected from 6 Kathiawari horses for its evaluation and freezing. The appearance of fresh semen was milky white to creamy and consistency of gel free semen was variably thin. The volume of total semen, gel free semen and gel in semen were 37.8±3.47, 25.8±2.13 and 12.0±2.22 ml, respectively. Mass and progressive sperm motility percent in gel free semen was 82.0±1.51 and 77.0±1.51, respectively. Mean sperm concentration in fresh semen was 173.75±8.86 × 106 ml-1. Post thaw sperm motility (PTM) % in 17 out of total 21 ejaculates (80%) was recorded up to an acceptable &gt;30 and mean value 35.62±1.99. Five Kathiawari horses were moderate to good freezer (PTM, 30 to 45%), whereas one stallion was poor freezer (PTM, 5 to 10%). The study indicated that the semen from Kathiawari stallions can be cryopreserved at farmers’ door using vapor freezing technique for encouraging artificial insemination and their conservation.

The Effects of Centrifuged Egg Yolk Used with INRA Plus Soybean Lecithin Extender on Semen Quality to Freeze Miniature Caspian Horse Semen

The aim of this study was to determine the synergistic effects of centrifuged egg yolk (EY) and soybean lecithin on post-thaw Caspian horse sperm motility, morphological abnormalities, and assessment of membrane integrity. The centrifuged EY (CEY) was added at concentrations of 2% and 4% to a defined INRA plus 1.25% soybean lecithin extender used to freeze Caspian horse semen. In this experiment, ejaculates collected from each Caspian horse (n = 4) were divided into three equal aliquots and diluted in CEY 2% (INRA2), 4% (INRA4) supplemented, and without any CEY (INRA0) in INRA plus 1.25% soybean lecithin extender, respectively. Thereafter, samples were frozen and thawed following a standard protocol. Sperm cryosurvival was evaluated in vitro by microscopy assessments of post-thaw sperm motility (by means of computer-assisted semen motility analysis [CASA]), acrosomal and other abnormalities (head, mid-pieces, and tail) and plasma membrane integrity (evaluated by HOST). In Caspian stallion, semen extended with INRA2 had significantly higher CASA motility and CASA progressive motility than those extended with the rest of extenders after freezing and thawing (P < .001). There was no significant difference in path velocity (VAP), VCL, and ALH among three groups (P > .05). For straight line velocity (P < .01) and LIN (P < .001), the highest values were obtained from the INRA4 group. The highest percentages of acrosomal and other abnormalities were found in semen diluted in INRA4 (P < .001). In the group frozen INRA2, the percentage of membrane integrity was significantly higher than that of the other groups (P < .001). The use of CEY 2% in combination with soybean lecithin significantly improved Caspian horse semen freezability.

Viability and fertility of cooled equine semen diluted with skimmed milk or glycine egg yolk-based extenders

Two semen extenders were compared for their ability to maintain viability of horse semen during 24 hours of cold preservation, and for the pregnancy rate after artificial insemination. In the experiment 1, five ejaculates from three stallions were split-diluted in either a skimmed milk-based extender (Kenney extender) or a glycine egg yolk-based extender (Foote extender) and cooled at 6-8 ºC for 24 hours. Semen samples stored in Kenney extender for 24 hours had higher motility and spermatic vigor compared with those stored in Foote extender. However, samples stored in Foote extender had higher number of reactive sperm by hypoosmotic test and greater viability by epifluorescence test compared with those in Kenney extender. In the experiment 2, 17 and 23 ejaculates from two stallions were split-diluted with Kenney extender and Foote extender. The sperm concentration in each extender was adjusted to 500 million viable sperms per insemination dose. Semen was cooled to 6-8 ºC and stored for 24 hours. Seventy-four cycles of crossbred mares were inseminated with either semen diluted in Kenney extender or semen diluted in Foote extender. The pregnancy rate was higher from semen diluted in Kenney extender than that from semen in Foote extender (0.553 vs. 0.306). The Kenney extender is effective in preserving the motility, vigor and fertility of stallion semen after 24 hours of cold storage, whereas the Foote extender is not acceptable.

Luteogenic and luteotropic effects of eCG during pregnancy in the mare

The role of eCG during pregnancy was evaluated through the study of the temporal relationships between changes in eCG and progesterone concentrations and the formation of supplementary corpora lutea (SCL) in mares impregnated with donkey semen (mule pregnancies) or with horse semen (equine pregnancies). Concentrations of eCG were higher (p<0.01) in equine than in mule pregnancies between weeks 6.5 and 13. Progesterone concentrations were higher in equine than in mule pregnancies between weeks 9 and 17. All animals developed at least one SCL, but more SCL accumulated during equine pregnancies than during mule pregnancies (1.9±0.2 vs 1.2±0.1; p<0.01). In equine pregnancies, the mares that formed a second SCL had higher eCG concentrations (p<0.05) during the two weeks preceding its formation than those mares remaining with only one SCL. Mares that formed a third SCL had higher (p<0.5) eCG levels than those remaining with one or two SCL. Mares with equine pregnancies that formed three SCL had higher progesterone concentrations (p<0.05) than those that formed only one or two SCL. No differences were found in progesterone or eCG concentrations between mares with mule pregnancies that accumulated different numbers of SCL during pregnancy (p>0.05). It is concluded that eCG stimulates both the development of new SCL and the function of existing CL. While these effects are clearly expressed in mares impregnated by horses, the low eCG concentrations during mule pregnancies reduce the impact of this hormone on CL formation and function.