Equine Artificial Insemination Research Articles

Rotation of liquid-preserved artificial insemination doses on roller benches affects sperm quality during storage in stallions.

Appropriate stallion semen handling is of great importance in equine artificial insemination (AI) industry. Optimal treatment of AI doses is aiming for best sperm preservation by excluding strong environmental influences and adding media that favour sperm survival. One method widely used in stallion sperm handling is the rotation of liquid-preserved semen samples on roller benches during storage. As previous studies in boars give rise to the fact that rotation should not be considered beneficial for spermatozoa anymore, the present study investigated the influence of continuous rotation of diluted stallion AI portions on sperm quality. Ejaculates (n = 15) were collected at a German AI centre and diluted with the two extenders EquiPlus and Gent (Minitüb GmbH) to a final concentration of 50 × 106 sperm/mL. Afterwards, samples were placed separately on roller benches at 5°C in the dark, obtaining a rotation frequency of 5 revolutions per minute (rpm) and 36 rpm for four consecutive days following a split-sample design. Both groups were analysed daily in comparison to a control group (0 rpm) with an extended spectrum of spermatological methods including computer-assisted sperm analysis and flow cytometry. Statistical analyses were based on the calculation of generalized linear mixed models for each sperm parameter. The research revealed a decrease in sperm quality parameters of rotated samples compared to non-rotated control groups, visible in total sperm motility (p < .001), decreased thermo-resistance (p < .01) and a drop in pH (p < .001). Interestingly, no differences (p > .05) were detected between rotation frequencies of 5 and 36 rpm. We conclude that the fertilizing capacity of stallion semen was negatively affected by rotation during storage in vitro, irrespective of the rotation frequency. Further studies need to investigate whether field fertility in horses is similarly affected by semen rotation on roller benches in vivo.

Evaluation of a portable device for assessment of motility in stallion semen.

ContentsIn horse breeding, quality assessment of semen before insemination is often requested. Non‐laboratory‐based techniques for objective analysis of sperm motility are thus of interest. The aim of this study was evaluating a portable device for semen analysis (Ongo sperm test) and its comparison with computer‐assisted semen analysis (CASA). Semen was collected from 10 stallions, diluted to 100, 50 and 25 × 106 sperm/ml and analysed for total (TM) and progressive motility (PM). The final sperm concentration influenced total motility analysed by Ongo (p < 0.05) which was higher at 100 × 106 sperm/ml when compared to 25 × 106 sperm/ml (p < 0.05) but not when compared to 50 × 106 sperm/ml (n.s.). Sperm concentration did not influence total motility when assessed by SpermVision (n.s.). Agreement between methods was evaluated by correlation analysis and Bland–Altman plot. Intra‐assay variation of Ongo was 5.2% ± 3.0 for TM and 6.9% ± 3.4 for PM. Correlation between Ongo and CASA was r = 0.79, 0.88 and 0.83 for 100, 50 and 25 × 106 sperm/ml for TM, and r = 0.87, 0.89 and 0.87 for PM, respectively (all p < 0.001). At the 100 and 25 mio/ml dilutions, the difference between the two systems deviated significantly from 0, while no such bias existed at the 50 mio/ml dilution (TM Ongo 85.0%, CASA 82.3%; PM Ongo 64.1%, CASA 66.1%). The 95% confidence interval was 19.9%, 18.9% and 19.2% ± mean for TM and 20.7%, 17.4% and 20.3% ± mean for 100, 50 and 25 × 106 sperm/ml, respectively. In conclusion, Ongo sperm test sperm motility data were strongly correlated with data obtained by CASA. In addition, at a concentration of 50 × 106 sperm/ml values measured with both systems were close to identical. At this concentration, which is recommended in equine AI, Ongo and CASA can be used interchangeably.

Use of frozen-thawed semen in equine artificial insemination programmes

Use of frozen-thawed semen in equine artificial insemination programmes

Practical guide to single layer centrifugation of stallion semen

SummaryThe technique of single layer centrifugation through species‐specific colloids has been gaining acceptance over the last few years as a practical method of improving the sperm quality of stallion semen for artificial insemination. This article explains the background to the technique and the different colloid formulations used, suggests some applications of the technique with particular reference to the equine breeding industry, describes the method in detail, and finally provides some guidance for problem solving. It is intended as a practical guide for those considering using the technique to improve sperm quality and pregnancy rates in equine artificial insemination and embryo transfer.

19 FACTORS AFFECTING PREGNANCY RATE AFTER ARTIFICIAL INSEMINATION WITH EQUINE SEMEN: PRODUCTION OF THE FIRST FOAL USING FROZEN SEMEN IN KOREA

Research in the area of equine artificial insemination (AI) has led to its increased application in field trials (Loomis and Graham, 2008). However, procedures for equine semen collection, cooling and freezing of semen and artificial insemination need further improvement. This study examined the effect of semen preparation and ovulation on equine AI success. Stallion semen was collected via artificial vagina, diluted with skim-milk extender, and preserved by either cooling to 4°C or cryopreservation and storage in liquid nitrogen. Mares were examined by ultrasound for follicle development with every 12 h during oestrus. During AI, semen was inserted into uterine horn adjacent to the ovulating follicle sized over 4 cm and irregular shape. In experiment 1, mares were inseminated with either cooled, cooled-transported, or frozen–thawed semen. Cooled semen was used immediately after collection and cooling. Cooled-transported semen was used 4 h after semen collection and dilution. Semen freezing procedure was followed by Moore et al. (2006). In experiment 2, we examined the effect of buffer (skim-milk extender), which was infused into the uterus at the time of AI with frozen–thawed semen. In experiment 3, we compared AI pregnancy rates for mares ovulating spontaneously v. after treatment with hCG. Pregnancy data were analysed Chi square test where P-values &lt; 0.05 were considered significantly different. In experiment 1, the pregnancy rates after AI using cooled, cooled-transported, and frozen–thawed semen were 60 (10/6), 50 (8/4), and 37.5% (8/3), respectively, and similar among treatments. In experiment 2, the pregnancy rate of mares infused with buffer at AI was 37.5% (8/3) which was higher than that with no buffer (0%; 6/0). In experiment 3, the pregnancy rates of mares were similar between ovulated spontaneously (25%; 8/2) and ovulated with hCG (50%; 8/4). The results suggest that equine semen that has been cooled, cooled and transported or frozen can be successfully used to establish AI pregnancy. Also, the pregnancy rates after AI can be increased by infusing buffer into the uterus at AI or by inducing ovulation with hCG. These experiments resulted in the first foal produced in Korea with frozen–thawed equine semen. This research was supported by Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

Dynamics of sperm DNA fragmentation in domestic animals

The mixed success of equine artificial insemination programs using chilled and frozen-thawed semen is most likely associated with the variable response of the sperm cell to the preservation process and the fact that stallions are not selected on the basis of reproductive performance. We propose that the traditional indicators of sperm viability do not fully account for male factor infertility in the stallion and that knowledge of sperm DNA damage in the original semen sample and during semen processing may provide a more informed explanation of an individual stallion's reproductive potential. This study reports on the validation of a sperm DNA fragmentation test based on the sperm chromatin dispersion test (SCD) for stallion spermatozoa and on its application to semen that was chilled (4 degrees C; n=10) or frozen-thawed (n=13). Semen samples were collected by artificial vagina and the proportion of sperm with fragmented DNA determined. Seminal plasma was then removed by centrifugation and the sperm pellet re-suspended in commercial extenders prior to being chilled or cryopreserved using standard industry protocols. Chilled semen was cooled slowly to 4 degrees C and stored for 1h before commencing the analysis; cryopreserved semen was thawed and immediately analyzed. Following chilling or cryopreservation, the semen samples were incubated at 37 degrees C and analyzed for SCD after 0, 4, 6, 24 and 48 h storage. The results of this investigation revealed that there was no significant difference in the sperm DNA fragmentation index (sDFI) of sperm evaluated initially after collection compared to those tested immediately after chilling or cryopreservation. However, within 1h of incubation at 37 degrees C, both chilled and frozen-thawed spermatozoa showed a significant increase in the proportion of sDFI; after 6h the sDFI had increased to over 50% and by 48 h, almost 100% of the sperm showed DNA damage. While the sDFI of individual stallions at equivalent times of incubation was variable, an analysis of the rate of change of sDFI revealed no difference between stallions or the way in which the semen was preserved. In terms of sperm DNA fragmentation dynamics, the highest intensity of sperm DNA damage occurred in the first 6h of incubation. We suggest that the SCD test can be used as a routine assessment tool for the development and refinement of preservation protocols designed to reduce stallion sperm DNA damage.

Retrospective study on the efficacy of hCG in an equine artificial insemination programme using frozen semen

Retrospective study on the efficacy of hCG in an equine artificial insemination programme using frozen semen

8. Equine artificial insemination in Iceland : G.Ö. Gudmundsson, District Veterinarian, Hvanncyri, IS-311 Borgarnes, Iceland

8. Equine artificial insemination in Iceland : G.Ö. Gudmundsson, District Veterinarian, Hvanncyri, IS-311 Borgarnes, Iceland

Equine Artificial Insemination

Artificial insemination is an effective technique for improving utilization of the stallion while maintaining normal conception rates in the mare. However, procedures for collection, evaluation, and insemination of semen must be followed carefully to achieve good results. Techniques for preservation of equine semen in the liquid or frozen state could potentially allow for more widespread use of genetically superior stallions. Further acceptance of artificial insemination and the use of cooled or frozen transported semen by breed registries is needed before this will occur. More work is needed to perfect methods of semen preservation, even though semen from some stallions can be cooled or frozen quite successfully at the present time.

Equine artificial insemination

The use and techniques of artificial insemination for horses in Germany over the last 30 years is described. Artificial insemination appears to produce pregnancy percentages equal to those from normal breeding methods and its continued availability under veterinary supervision is recommended in conditions where disease, disability or distance debar normal service.