Insemination Of Mares Research Articles

A simplified fixed-time insemination protocol using frozen-thawed stallion spermatozoa stored at 17°C for up to 24 h before insemination.

Insemination of mares with frozen-thawed spermatozoa requires intensive management and results in 40%-60% per cycle pregnancy rates. To determine if satisfactory fertility is possible for frozen-thawed semen after processing it through a microfluidic device, followed by storage at 17°C for up to 24 h before fixed-time insemination. Uncontrolled field trials. A pilot study evaluated the motility of frozen-thawed spermatozoa after centrifugation and storage (17°C) in two different media for up to 48 h. Subsequently, the motility of frozen-thawed semen processed through a microfluidic device, resuspended in two different media during storage (17°C) for up to 24 h was evaluated. The fertility of frozen-thawed spermatozoa, after microfluidic sorting and storage at 17°C for up to 24 h, was evaluated after fixed-time insemination in a commercial embryo programme. Experiment 1: Frozen-thawed spermatozoa (N = 5 stallions) were centrifuged and resuspended in Botusemen Gold™ or SpermSafe™ and stored (17°C) for up to 48 h. Sperm motility was evaluated by CASA at 0, 6, 24 and 48 h. Experiment 2: Frozen-thawed spermatozoa (N = 4 stallions) underwent microfluidic sorting and storage (17°C) for up to 24 h in both media. Sperm concentration and motility were evaluated at 0, 16 and 24 h. Experiment 3: Fertility of frozen-thawed spermatozoa (N = 3 stallions) was evaluated after insemination of 42 mare cycles at 6, 16 and 24 h after thawing, microfluidic sorting and storage before fixed-time insemination. The stallion significantly influenced sperm motility, but there was no effect of media on motility parameters. Storage time significantly affected sperm motility after centrifugation but not after microfluidic sorting. Storage time had no effect on the overall embryo recovery rate (52%, n = 42). Field trial with small mare numbers and no control at time = 0 h. Fixed-time insemination of frozen-thawed spermatozoa after microfluidic sorting and storage at 17°C for up to 24 h produced satisfactory embryo recovery rates.

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).

Predicting the Outcome of Equine Artificial Inseminations Using Chilled Semen

This study aimed to determine whether an analysis of stallion ejaculate could accurately predict the likelihood of pregnancy resulting from artificial insemination in mares. This study involved 46 inseminations of 41 mares, using 7 standardbred stallions over a 5-week period at an Australian pacing stud. Semen quality was assessed immediately after collection and again after chilling at ~5 °C for 24 h. The assessment involved evaluating ejaculate volume, sperm concentration, and motility parameters using an iSperm® Equine portable device. After the initial evaluation, a subpopulation of cells was subjected to a migration assay through a 5 µm polycarbonate filter within a Samson™ isolation chamber over a 15 min period. The cells were assessed for their concentration, motility parameters, and ability to reduce the membrane impermeant tetrazolium salt WST-1. The data, combined with the stallion and mare's ages, were used to predict the likelihood of pregnancy, as confirmed by rectal ultrasound sonography performed 14 days post ovulation. The criteria used to predict pregnancy were optimized for each individual stallion, resulting in an overall accuracy of 87.9% if analyzed pre-chilling and 95% if analyzed post-chilling. This study suggests that an analysis of stallion ejaculate can be used to predict the likelihood of pregnancy resulting from artificial insemination in mares with a high level of accuracy.

L-carnitine added to post-thawed semen acts as an antioxidant and a stimulator of equine sperm metabolism.

The objective of this study was to enhance the in vitro sperm quality and in vivo fertility of frozen-thawed equine semen by the addition of l-carnitine (LC) to post-thawed semen. Different concentrations of LC were added to thawed samples to obtain four treatments control and 0.5, 1 and 2mM LC. In the in vitro experiments, sperm motility and kinematics, membrane integrity and intracellular calcium ion concentration ([Ca2+ ]i ) were investigated, and the antioxidant bioactivity of LC was assessed by measuring hydrogen peroxide and nitrite concentrations (NO2 - ). The fertility rate was assessed via the artificial insemination of mares. The treatment with 1mM LC increased sperm [Ca2+ ]i (60.6±0.05 AU), reduced nitrite concentration (39.1±14.9µM/µg protein), increased the sperm straightness percentage (STR: 78.3±5.3%) and increased the pregnancy rate (75%) as compared to the control ([Ca2+ ]i 48.4±0.05 AU, NO2 - concentration 63.1±14.4µM/µg protein, STR 67.5±7.9%, 12.5% pregnancy rate, p<0.05). These results suggest that 1mM LC acts as an antioxidant and stimulator of sperm metabolism in post-thawed equine semen, increasing the fertility rate. Thus, addition of LC might be an alternative to improve the fertility of poor quality post-thawed equine semen.

Two hormonal protocols for timed artificial insemination in mares under different residence times of the progesterone intravaginal device

Two hormonal protocols for timed artificial insemination in mares under different residence times of the progesterone intravaginal device

LCCY (lactose-chelate-citrate-yolk ) environment - the key to success in insemination of mares with chilled semen stored for up to 5 days

LCCY (lactose-chelate-citrate-yolk ) environment - the key to success in insemination of mares with chilled semen stored for up to 5 days

The effectiveness of artificial insemination in horse breeding

As a result of achievements in genetics and in technology of artificial insemination` cattle breeders received a powerful instrument of improving animals productive qualities, the possibilities of selecting stallions with a high genetic potential of productivity were expanded` and the rate of genetic improvement of entire populations was accelerated. Sperm from stallions is taken on an artificial vagina. Evaluation of the quality of sperm is carried out for 3 consecutive days. Insemination of mares is carried out at artificial insemination stations. 10 days after ovulation the mares are checked again with a probing stallion. If the mare for 35–40 days has not shown signs of sexual hunting again then it is followed by rectal test for foal.

АНТИГЕННЫЙ СОСТАВ СИСТЕМ ГРУПП КРОВИ И КАЧЕСТВО СПЕРМЫ ЖЕРЕБЦОВ РАЗНЫХ ПОРОД

Artificial insemination of mares with cryopreserved sperm is quite widespread in horse breeding practice and contributes to the efficiency of genetic improvement and preservation of breeds. The purpose of our research was to study the associations of erythrocyte antigen systems with the quality of stallions' sperm before and after freezing, taking into account their breed affiliation. Our studies evaluated the sperm production of 141 stallions tested on A, C, D, and K blood group systems that included 13 red blood cell antigens (Aa, Ad, Ca, Da, Db, Dc, Dd, De, Dg, Dh, Dk, Dm, and Ka). The evaluation of cryopreserved sperm was performed based on the average indicators of sperm activity in points and survival time in hours. The mobility of the newly obtained sperm of stallions varied in the range of 1.2-8.5 points with an average value of 4.45 points and minor differences between representatives of different breed types. The best indicators of activity (2.24 points) and survival (77 hours) of cryopreserved sperm were stallions of domestic heavy-strain breeds. The influence of a system of blood groups (F=3,023, P=0,006) on the activity of fresh sperm was revealed. The relatively good quality of cryopreserved sperm was distinguished by sires of riding breeds with the presence of Dc, Dg and Dm, Orlov Trotter stallions with Ca, K - , Dg and Dk, as well as representatives of prize and heavy-duty breeds with Dd. The results obtained show that specific gene complexes and associations with productive traits are formed in horse breeds of different directions under the influence of selection, which should be taken into account in the methodology of scientific research.

Reproductive Characteristics of Thawed Stallion Sperm.

Simple SummaryIt is very important to evaluate correctly the fertilizing capacity of frozen sperm before carrying out the artificial insemination of mares. It is not always possible to perform complex laboratory tests under the stud farm’s conditions. The purpose of our study was to determine the minimum set of relatively simple laboratory tests sufficient to confirm the feasibility of frozen/thawed sperm for insemination and to predict the pregnancy rate in mares. We found that sperm characteristics such as activity, survival at low temperature, and DNA damage are sufficient to determine the quality of frozen/thawed sperm. These characteristics can be determined by simple tests and assays that can be used for increasing the effectiveness of artificial insemination not only in horse breeding but also in other areas of livestock and even in reproductive medicine.The main goal of our study was to determine a set of thawed stallion sperm characteristics that have predictive value for the pregnancy rate (PR) of mares after artificial insemination (AI). DNA fragmentation and survival of sperm during hypothermic storage were studied in addition to routinely determined semen characteristics such as concentration, percentage of motile spermatozoa, and morphology. To estimate DNA fragmentation, a modified hallo assay was applied. Sperm survival was determined within hours as the ability of spermatozoa to maintain progressive motility (PM) during the storage of ejaculate diluted with lactose-chelate-citrate-yolk (LCCY) medium at +4 °C. Strong positive correlation between PR and thawed sperm motility (r = 0.90, p < 0.05) as well as between PR and sperm survival (r = 084, p < 0.05) was revealed. There was also a strong negative correlation between PR and DNA damages in spermatozoa (r = −0.94, p < 0.05). We found no dependence of PR on normal morphology spermatozoa percentage in thawed semen. We concluded that the sperm activity, survival, and DNA fragmentation should be considered as the sufficient reproductive characteristics of semen to evaluate the quality of frozen/thawed sperm and prediction of PR.

Factors Influencing the Popularity of Artificial Insemination of Mares in Europe.

Simple SummaryThe popularity of mare insemination as an element affecting the dynamic growth of breeding progress among horses in Europe is subject to various fluctuations. The success of this method of reproduction should be considered first of all in terms of the quality of semen available on the market, the types of semen storage technology, the profitability of its use resulting from the effectiveness of insemination with the selected type of semen, and factors affecting the success of artificial insemination. The purpose of this work was to present the factors determining the popularity of artificial insemination of mares in Europe. The presented statistics show that the popularity of the use of chilled semen has gradually increased in the group of sport mares, while in the group of breeding mares, the popularity of frozen semen has increased. In the remaining group of mares (not classified as sport or breeding), insemination with chilled semen was dominant. To talk about the success of artificial insemination of horses in Europe, it is necessary to look thoroughly at these aspects that affect the popularity of this reproduction biotechnology, and in particular to improve the quality of insemination doses.The purpose of this review was to analyze factors affecting the popularity of artificial insemination of mares in Europe in the context of sperm quality. Taking into account the prices of stallion semen on the world market, efficiency is important for the profitability of its use in artificial insemination programs in Europe. To increase the efficiency of a semen insemination facility, it is necessary to correctly and objectively assess the quality of semen. The available range of tools allows an effective evaluation of the potential fertility of a stallion. For several years, artificial insemination programs in Europe have been gaining popularity. However, the frequency of chilled or frozen semen use is still quite low. This is mainly due to the common, negative opinion about the effectiveness of the use of packaged insemination doses as opposed to natural insemination. Unfortunately, the quality of the semen offered often deviates from expectations, which results in unsatisfactory (and therefore unprofitable) pregnancy rates. This review presents the popularity structure of chilled and frozen semen use in European horse breeding as well as the current state of research on the effectiveness of semen production technology. It is shown that the popularity of using chilled semen in the artificial insemination of mares in Europe has been gradually increasing in the group of sport mares, while in the group of breeding mares, in recent years, frozen semen has been gaining popularity. In the remaining group of mares (not classified as sport or breeding), insemination with chilled semen has been dominant.

Uterine Immune Response After Single and Double Fresh Sperm Insemination in Mares

The aim of the study was to evaluate the effects of single and double inseminations on the inflammation of the uterus in mares. Nine mix breed mares with endometrial biopsy scores grade I and IIA, aged 5-15, were used in the study. Two experiments were performed over two estrous cycles: (1) single insemination, (2) double insemination. All mares were used in both insemination group. In the single insemination cycles one insemination was performed 24 h after hCG injection, while in the double insemination cycles two inseminations were carried out 24 h and 48 h after the hCG injection. Endometrial biopsies were collected in both cycles immediately before (=0), and 6, 30 and 54 h after the first insemination. Gene expressions of the inflammatory markers IL-1β, IL-6, IL-8, iNOS, SAA, COX-2 and CASP-3 and histopathological alterations of the endometrium were determined. The 6 h expression levels of IL-6 were higher when compared with 30 h and 54 h (P<0.05). CASP-3 expression levels in single insemination cycles were higher at 0 h and 6 h when compared with 54 h (P<0.05). In conclusion expression levels of the IL-1β,-6,-8, iNOS, SAA and COX-2 were not different between single or double insemination cycles. This study provides preliminary evidence to further characterize the changes in the expression of relevant genes in response to fresh semen.

Influence of artificial insemination time period relative to ovulation on fertility of mares

The results of the study of the effect of artificial insemination of horses by the cooled and thawed semen which was prepared by Kharkiv technology depending on the time of the dominant follicle ovulation using the atraumatic tool developed for insemination of mares have been presented in the article. In modern practice of artificial insemination of horses, there are two opposing views on the timing of insemination of mares (before or after ovulation) so we decided to investigate the impact of this factor on sperm, obtained, chilled and kriokonse–rvovana for Kharkiv technology. The time of semen administration into the reproductive tract of mares depending on the ovulation of the dominant follicle is the least investigated factor that effects the efficiency of mare insemination. The artificial insemination of the horses by the semen that was prepared by Kharkiv technology with the use of the atraumatic instrument developed by us for artificial insemination of mares depending on the time of ovulation of the dominant follicle was conducted for the first time. It has been stated that the optimal time for the insemination of mares by the cooled or thawed semen of stallions depending on the ovulation with the use of the atraumatic instrument developed by us is the time period for 4 hours before and after ovulation. When insemination of mares Ukrainian horse breed tools we have and the sperm that chilled by Kharkivtion technology for 4 hours before and after ovulation received about the same output foals. When artificial insemination Ukrainian horse breed mares we developed noninvasive device after 4 or more hours of ovulation foals output decreased by 50.77% (P &lt; 0.01) compared with insemination to ovulation and the 50.12% (P &lt; 0.01) compared with insemination after ovulation. When artificial insemination of mares Ukrainian horse breed tools we have and sperm cryopreserved for that Kharkov Technologies for 4 hours after ovulation received incredibly more foals out at 3.47% compared with insemination before ovulation. When artificial insemination of mares Ukrainian horse breed developed our noninvasive device after 4 or more hours of ovulation foals output decreased by 35.23% (P &lt; 0.01) compared with insemination to ovulation and the 38.70% (P &lt; 0,01) compared to the insemination after ovulation. When carrying out the artificial insemination of the mares of Ukrainian horse breed for more than 4 hours after the ovulation the fertility decreased by 50,12 – 50.77% (P &lt; 0.01) and by 35,23 – 38,70% (P &lt; 0.01) by cooled and thawed semen, respectively.

Effect of Multiple Freezing of Stallion Semen on Sperm Quality and Fertility

To increase the efficiency of equine semen, it could be useful to split the artificial insemination dose and refreeze the redundant spermatozoa. In experiment I, semen of 10 sires of the Hanoverian breed, with poor and good semen freezability, was collected by artificial vagina, centrifuged, extended in INRA82 at 400 × 106 sperm/mL, and automatically frozen. After this first routinely applied freezing program, semen from each stallion was thawed, resuspended in INRA82 at 40 × 106 sperm/mL, filled in 0.5-mL straws, and refrozen. These steps were repeated, and sperm concentration was adjusted to 20 × 106 sperm/mL after a third freezing cycle. Regardless of stallion freezability group, sperm motility and sperm membrane integrity (FITC/PNA-Syto-PI-stain) decreased stepwise after first, second, and third freezing (62.3% ± 9.35, 24.0% ± 15.4, 3.3% ± 4.34, P ≤ .05; 29.6% ± 8.64, 14.9% ± 6.38, 8.3% ± 3.24, P ≤ .05), whereas the percentage of acrosome-reacted cells increased (19.5% ± 7.59, 23.9% ± 8.51, 29.2% ± 6.58, P ≤ .05). Sperm chromatin integrity was unaffected after multiple freeze/thaw cycles (DFI value: 18.6% ± 6.6, 17.2% ± 6.84, 17.1% ± 7.21, P > .05). In experiment II estrous, Hanoverian warmblood mares were inseminated with a total of 200 × 106 spermatozoa of two stallions with either good or poor semen freezability originating from the first, second, and third freeze/thaw cycle. First-cycle pregnancy rates were 4/10, 40%; 1/10, 10%; and 0/10, 0%. In conclusion, as expected, sperm viability of stallion spermatozoa significantly decreases as a consequence of multiple freezing. However, sperm chromatin integrity was not affected. Pregnancy rates after insemination of mares with refrozen semen are reduced.

Colloid Centrifugation of Semen: Applications in Assisted Reproduction

Colloid centrifugation can be used to separate heterogeneous populations of cells particularly semen samples, which typically contain mixtures of spermatozoa at different stage of maturity together with dead and dying spermatozoa and also non-sperm cells. Colloids can be used to enrich the population of spermatozoa that are viable and functional; these are the spermatozoa that are needed for fertilization in Assisted Reproduction. Sperm samples obtained after colloid centrifugation may be enriched for motile, morphologically normal spermatozoa with intact chromatin. Insemination of mares with stallion sperm samples prepared by colloid centrifugation resulted in a higher pregnancy rate per cycle than controls, confirming that the good sperm attributes observed in the laboratory were indicative of functional spermatozoa. The method does not simply enrich for viable spermatozoa by inhibiting dead or dying spermatozoa from passing through the colloid since emerging evidence shows that colloid centrifugation can select for spermatozoa with certain properties such as metabolic activity. Other reproductive cells, such as spermatids or progenitor cells, may also be purified by colloid centrifugation. This review describes the history of colloid centrifugation for selecting spermatozoa, compares colloid centrifugation with alternative techniques, and finally describes some of the applications of the technique within the animal breeding industry.

14 EPIGENETIC CHANGES IN EQUINE SPERMATOZOA DURING CRYOPRESERVATION: A PRELIMINARY STUDY

The use of cryopreserved semen for insemination of mares facilitates breeding management but often results in reduced conception rates. This has been mainly attributed to changes in sperm membrane function caused by the freezing-thawing procedure. However, semen processing may also contribute to epigenetic changes in spermatozoa. In the present study, we therefore addressed changes in sperm DNA-methylation induced by cryopreservation of stallion semen. We hypothesised that the cryoprotectant may influence the DNA-methylation level of frozen-thawed semen. For this purpose, semen was collected from fertile Shetland pony stallions. Global DNA-methylation was assessed by ELISA (5-mC DNA ELISA Kit, Zymo Research, Irvine, CA, USA) with a monoclonal antibody sensitive and specific for 5-methylcytosin after DNA extraction and denaturation (100 ng of DNA per sample). The level of 5-methylcytosin in DNA is reported as the amount of methylated cytosine relative to the cytosine genomic content (%). Statistical analysis was done with the SPSS Statistics 21 software. Values are means ± standard error of the mean. In Experiment 1, 1.5 mL of raw semen (n = 6 stallions, 1 ejaculate each) was shock-frozen at –196°C for 15 min and thawed at 38°C for 60 s. Semen motility and membrane integrity were completely absent, while DNA-methylation was similar in raw (0.4 ± 0.2%) and shock-frozen (0.3 ± 0.1%) semen (not significant). In Experiment 2, 3 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 (Minitube, Tiefenbach, Germany; cryoprotectant: 5% glycerol) or BotuCrio (Nidacon, Mölndal, Sweden; cryoprotectants: 1% glycerol and 4% methylformamid) extender. Semen was frozen in 0.5-mL straws in a computer-controlled rate freezer (IceCube 14 M; Sylab, Purkersdorf, Austria, cooling rates: 20°C to 5°C: 0.3°C min–1, 5°C to 25°C: 10°C min–1, –25°C to –140°C: 25°C min–1). Semen motility, morphology, and membrane integrity were significantly reduced (e.g. total motility before freezing: 88.8 ± 1.4%) by cryopreservation but not influenced by the extender used (e.g. total motility: Ghent 69.5 ± 2.0, BotuCrio 68.4 ± 2.2%; P &lt; 0.001 v. nonfrozen semen). Cryopreservation significantly (P &lt; 0.01) increased the level of DNA-methylation (before freezing: 0.6 ± 0.1%, Ghent 6.4 ± 3.7, BotuCrio 4.4 ± 1.5%; P &lt; 0.01), but no differences between the freezing extenders were seen. The level of DNA-methylation was not correlated with semen motility, morphology, or membrane integrity. The results demonstrate that semen processing for cryopreservation increases the DNA-methylation level in stallion semen. In the present study, this effect occurred irrespective of the cryoprotectant but was not seen after shock-freezing in the absence of cryoprotectants. The reduced fertility of mares after insemination with frozen-thawed semen may at least in part be explained by methylation of sperm DNA, which occurs in response to the cryopreservation procedure.

Influence of the uterine inflammatory response after insemination with frozen–thawed semen on serum concentrations of acute phase proteins in mares

The aim of this study was to investigate the clinical relevance of measuring blood concentrations of serum amyloid A (SAA), haptoglobin (Hp) and fibrinogen (Fib) in horse reproductive management, and changes in response to artificial insemination (AI) with frozen–thawed semen. Standardbred mares (n=18) with different reproductive status (eight healthy mares in first postpartum oestrus, five healthy barren mares and five mares with endometritis) were inseminated with frozen–thawed semen. Endometritis was evaluated during oestrus by bacteriological culture, cytology and presence of ultrasonically visible intrauterine fluid during oestrus. Concentrations of SAA, Hp and Fib were analysed in the blood in every 48h during oestrus and until 5, 6 or 7 days after AI. The day of sampling and number of blood samples varied between mares because of length of the oestrus and time of AI. Changes in concentrations of SAA, Hp and Fib were evaluated based on the day of sampling regard to AI and classification of the mares. There were no differences in SAA, Hp and Fib concentrations over time before or after AI or between the groups of mares. The insemination of mares with frozen–thawed semen did not increase the plasma concentrations of SAA, Hp and Fib above clinical threshold concentration and there were no differences between susceptible or healthy mares.

Factors affecting the uterine inflammatory response to semen

At the time of breeding or insemination the uterus is exposed to seminal components, bacteria and debris. This results in an inflammatory response characterized by the influx of polymorphonuclear neutrophils (PMN) into the uterine lumen. (1) The purpose of the inflammation is to clear from the uterus defective and excess sperm, and other contaminating agents. The influx of PMNs into the uterine lumen results in phagocytosis of sperm, bacteria and release of prostaglandin, which causes uterine contractions. (2) This inflammation is quickly cleared from the uterus within 36 hours. In fact, in most mares that are resistant to delayed post breeding endometritis, the uterus is clear of fluid by 24 hours with the peak of PMNs and uterine fluid occurring at 12 hours after insemination. It has often been stated that insemination of mares with frozen semen results in a greater inflammatory response However there are limited data on the response of the mare`s uterus to various types of semen. This manuscript addresses some of the factors that affect the severity of the inflammatory response after breeding.

Effect of oxytocin and flunixin meglumine on uterine response to insemination in mares

The most probable reason for persistent postbreeding endometritis in mares is weak myometrial contractility. The influence of oxytocin (OT; an ecbolic agent) and flunixin meglumine (FLU; a prostaglandin inhibitor serving as a model for mares with decreased uterine contractility) on uterine response to artificial insemination (AI) was studied in mares with no history of reproductive failure. The mares were treated intravenously with 10 mL saline (Group C, n = 10) or 0.01 IU/kg OT (Group OT, n = 10) 2, 4, 8, and 25 h after AI. Group FLU (n = 11) was treated with 1.1 mg/kg FLU 2 h after AI and with saline thereafter. The mares received the same treatments in the first and third cycles but were sampled either at 8 or 25 h. The amount of intrauterine fluid (IUF) and edema and the number of uterine contractions were recorded before AI and 10 min after the treatments using transrectal ultrasonography. At 8 h after AI, the mares were treated with human chorionic gonadotropin, and, after 8-h or 25-h scans, a 500-mL uterine lavage and a biopsy were performed. Ovulation was confirmed at 48 h and pregnancy 14 to 17 d after AI. No manipulations were done during the second estrus. At 8 h after AI, Group FLU had more polymorphonuclear leukocytes (PMNs) in the uterine lavage fluid than did Group OT (P < 0.05), but uterine contractions did not differ significantly. At 25 h, the PMN concentrations were low in all groups. Group OT rarely showed IUF. The uterine biopsy specimens of Group FLU showed less inflammation of the stroma but more PMNs in the uterine lumen 8 h after AI than that of the control group (P < 0.05). The pregnancy rates did not differ between the groups (63% C, 53% OT, and 50% FLU). Oxytocin rapidly and effectively removed IUF and PMNs after AI and thereby shortened the duration of postbreeding inflammation.

Embryo recovery following artificial insemination of mares enrolled in a small AI breeding program

In Portugal, there is much interest in the so-called assisted reproductive technologies as applied to horse breeding, namely at the semen cryopreservation and Artificial Insemination levels. The interest for embryo transfer in mares has also emerged despite several problems associated with this technique, such as the resistance of most breed registration authorities in accepting progeny begot by this method. The intention of this study was to evaluate the accuracy of embryo recovery techniques at a small facility and to consider the feasibility of its implementation for embryo transfer. Seven mares were alternatively inseminated in 13 successive oestrous cycles with fresh semen from 3 stallions of varying fertility. Ovulation was left to occur spontaneously, and embryo recovery was carried out by non-surgical uterine flushing using both a closed flushing system (RS1) and an open flushing system (RS2). A total of 13 embryo collection attempts were performed on 7 possible positive pregnancy diagnoses with a rate success of 57.14% (4/7). Our preliminary results showed that the open flushing recovery system gives the best results and the high success recovery rate suggests that the technique of embryo transfer can be fully implemented even in facilities with limited resources.

The efficient use of equine cryopreserved semen

In order to optimize the efficient use of cryopreserved stallion semen, recent research has focused on the minimum insemination dose of frozen–thawed spermatozoa required for maximum fertility rate. The results appear to be highly stallion-dependent. Factors such as the timing of AI with respect to ovulation, as well as the site of insemination within the mare's reproductive tract, also affect success in breeding with frozen–thawed semen. Since acceptable pregnancy rates can be achieved from insemination of mares with very low numbers of spermatozoa, increasing the number of insemination doses processed from a single ejaculate may prove more cost-effective to stallion owners.