Hysteroscopic insemination of low numbers of flow sorted fresh and frozen/thawed stallion spermatozoa.
Abstract: The objective of this experiment was to determine the effects of flow cytometric sorting and freezing on stallion sperm fertility. A 2 x 2 factorial design was used to delineate effects of flow sorting and freezing spermatozoa. Oestrus was synchronised (July-August) in 41 mares by administering 10 ml altrenogest (2.2 mg/ml) per os for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. Ovulation was induced by administering 3,000 iu hCG i.v. either 6 h (fresh spermatozoa) or 30 h (frozen/thawed spermatozoa) prior to insemination. Mares were assigned randomly to one of 4 sperm treatment groups. Semen was collected from 2 stallions with an artificial vagina and processed for each treatment. Treatment 1 (n = 10 mare cycles) consisted of fresh, nonsorted spermatozoa and Treatment 2 (n = 16 mare cycles) of fresh, flow sorted spermatozoa. Spermatozoa to be sorted were stained with Hoechst 33342 and sorted into X- and Y-chromosome-bearing populations based on DNA content using an SX MoFlo sperm sorter. Treatment 3 (n = 16 mare cycles) consisted of frozen/thawed nonsorted spermatozoa (frozen at 33.5 x 106 sperm/ml in 0.25 ml straws) and Treatment 4 (n = 15 mare cycles) of flow sorted frozen/thawed spermatozoa (frozen at 64.4 x 10(6) sperm/ml). Concentrations of sperm in both cryopreserved treatments were adjusted, based on predetermined average post-thaw motilities, so that each insemination contained approximately 5 x 10(6) motile spermatozoa. Hysteroscopic insemination of 5 x 10(6) motile spermatozoa in a volume of 230 microd was used for all treatments. Pregnancy was determined ultrasonographically 16 days postovulation. No differences were found (P>0.1) in the pregnancy rates for mares inseminated with fresh nonsorted (4/10 = 40.0%), fresh flow sorted (6/16 = 37.5%), frozen/thawed nonsorted (6/16 = 37.5%) and flow sorted frozen/thawed spermatozoa (2/15 = 133%). Pregnancy rates tended (P = 0.12) to be lower following insemination of frozen/thawed flow sorted spermatozoa. Further studies are needed with a larger number of mares to determine if fertility of flow sorted frozen/thawed spermatozoa can be improved.
Publication Date: 2002-03-21 PubMed ID: 11902755DOI: 10.2746/042516402776767321Google Scholar: Lookup
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- Journal Article
Summary
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The research was conducted to study the impact of certain processes – flow cytometric sorting and freezing – on the fertility of stallion sperm. The study found no significant difference in pregnancy rates when using fresh or frozen sperm, whether it had been sorted or not. However, further research is suggested to verify if the fertility of flow sorted frozen sperm can be enhanced.
Objective and Methodology
- The experiment aimed at understanding the effects of flow cytometric sorting (a method used to sort or separate microscopic particles, in this case, spermatozoa) and freezing on stallion sperm fertility.
- The study employed a 2 x 2 factorial design. This means they used two types of sperm (fresh and frozen) and two treatments (sorted and unsorted) to examine the four combinations for their potential impact on fertility.
- The trial began with the synchronization of oestrus in 41 mares, inducing ovulation at specific times before insemination.
- The mares were then randomly assigned to one of the four treatment groups, fresh non-sorted, fresh flow sorted, frozen/thawed nonsorted, and frozen/thawed flow sorted spermatozoa.
Conducting the Experiment
- Semen was collected from two stallions and processed to delineate the effects of flow sorting and freezing spermatozoa.
- The flow sorting involved staining the collected sperm with a particular dye (Hoechst 33342) and segregating them into X and Y chromosome-bearing populations. This was done using an SX MoFlo sperm sorter.
- In treatments involving frozen spermatozoa, the sperm was frozen at specific concentrations.
- Regardless of the treatment, the concentration of the inseminated sperm was adjusted so that each insemination contained approximately five million motile spermatozoa.
Results and Conclusion
- Pregnancy in the mares was determined ultrasonographically (using ultrasound), 16 days post ovulation.
- The study’s findings showed no significant differences in the pregnancy rates among mares inseminated with the four different treatments of spermatozoa.
- The data did suggest, however, that the pregnancy rates were slightly lower following the insemination of frozen/thawed flow sorted spermatozoa. But this difference wasn’t statistically significant (P>0.1).
- The researchers concluded that more investigations are necessary with a larger sample of mares to determine if the fertility of flow sorted frozen/thawed spermatozoa can be improved.
Cite This Article
APA
Lindsey AC, Schenk JL, Graham JK, Bruemmer JE, Squires EL.
(2002).
Hysteroscopic insemination of low numbers of flow sorted fresh and frozen/thawed stallion spermatozoa.
Equine Vet J, 34(2), 121-127.
https://doi.org/10.2746/042516402776767321 Publication
Researcher Affiliations
- Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, USA.
MeSH Terms
- Animals
- Cryopreservation / veterinary
- Female
- Fertility / physiology
- Flow Cytometry / veterinary
- Freezing
- Horses / physiology
- Hysteroscopy / veterinary
- Insemination, Artificial / methods
- Insemination, Artificial / veterinary
- Male
- Pregnancy
- Pregnancy Rate
- Random Allocation
- Semen Preservation / veterinary
- Spermatozoa / physiology
Citations
This article has been cited 4 times.- Cherouveim P, Vagios S, Hammer K, Fitz V, Jiang VS, Dimitriadis I, Sacha CR, James KE, Bormann CL, Souter I. The impact of cryopreserved sperm on intrauterine insemination outcomes: is frozen as good as fresh?. Front Reprod Health 2023;5:1181751.
- Orsolini MF, Meyers SA, Dini P. An Update on Semen Physiology, Technologies, and Selection Techniques for the Advancement of In Vitro Equine Embryo Production: Section II. Animals (Basel) 2021 Nov 20;11(11).
- Kowalczyk A, Czerniawska-Piątkowska E, Kuczaj M. Factors Influencing the Popularity of Artificial Insemination of Mares in Europe. Animals (Basel) 2019 Jul 19;9(7).
- Quelhas J, Pinto-Pinho P, Lopes G, Rocha A, Pinto-Leite R, Fardilha M, Colaço B. Sustainable animal production: exploring the benefits of sperm sexing technologies in addressing critical industry challenges. Front Vet Sci 2023;10:1181659.
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