Cryopreservation of Equine Semen Loaded in Cryovials

Ano: 2011 | Volume: 39 | Número: 2

Autores: Sabrina Leães Gomez Lorenzoni, Natália Schmidt Arruda, José Luiz Rodrigues

Palavras-chave: sêmen, diluents, equine, freezing techniques, cryopreservation, cryovial

Resumo Inglês:

Background: Over the past twenty years the assisted reproduction techniques reached a rapid advance in domestic species of
economic interest. By the time the male gamete has provided to be the most successful tool used to improve animal breeding
programs. Freezing and stock of semen is a safe procedure to preserve reproductive potential of animals with superior genetic
heritage. In the horse industry, unlike observed in ruminants, the development of sperm cryopreservation techniques is very
slow but despite the technical barriers the artificial insemination with frozen semen is growing.
Materials, Methods & Results: This experiment was divided in three steps. The first one compared the use of different diluents
and freezing techniques. The other two stages were the use of cryogenic tubes for storing semen and the determination of mare
pregnancy rates that were artificially inseminated with frozen semen packaged in cryogenic tubes. Six stallions were submitted
to semen collection and twelve mares were artificially inseminated at first estrus of the breeding season. The samples were
diluted in INRA82 or Nagase, containing 5% glycerol, with a concentration of 200 x 106 sperm/mL. A fraction of the diluted
semen was stored in straws (0.5 mL), some of those straws were immediately frozen. The remaining straws were cooled from
room temperature (± 24°C) to 5°C before freezing. The samples were thawed in a water bath at 37°C during 30 s. The same
protocol with cooling prior to freezing was performed with samples filled into cryogenic tubes, and these samples were thawed
in a water bath at 50°C during 100 s. The in vivo efficiency of cryopreserved semen was determined through artificial
insemination (AI). The pregnancy diagnosis was performed after 20 days by ultrasound examination. After analyzing the data
it was found that there was no significant difference in sperm motility and vigor among the tested diluents. Differences (P =
0.05) were observed when the diluted semen was previously cooled or frozen directly. Sperm motility obtained with semen
diluted into INRA82 was 43.32% and 26.66%, and using Nagase solution the sperm motility was 41.08% and 24.44%,
respectively. There were no differences regarding the vigor, motility and sperm defects among the diluents and the semen
containers. The mares inseminated with semen loaded in cryovials showed 50% (3/6) pregnancy rate, and 16.66% (1/6) was
observed in the mares inseminated with semen loaded in straws. All pregnant mares delivered health foals.
Discussion: The fertilizing capacity of frozen semen is influenced by several factors including stallion, semen characteristics,
freezing technique, insemination dose, as well as status and management of the mare. Glycerol is the most commonly
cryoprotectant used to preserve equine spermatozoa. We first investigate the efficiency of two semen extenders in promoting
the survival of frozen spermatozoa. After that the use of two cryopreservation procedures, one cooling previously the semen
samples at 5°C before freezing and the other by exposing the semen directly to liquid nitrogen vapor. The statistic analysis
revealed no differences among the tested cryoprotectant solutions in provide sperm survival assessed by motility and vigor.
Moreover, comparison of tested cryopreservation procedures revealed significant differences in sperm survival rates, taking
into account both used semen extenders (INRA82 or Nagase). The fertilizing capacity of cryopreserved semen was checked by
the AI of mares at the first estrus of the breeding season, with only one insemination performed close to ovulation. The sperm
motility rates were higher when the semen was previously cooled, independent of the used semen extender. The survival rate
of sperm samples loaded into straws or cryovials was similar. The semen stored in cryovials is a viable alternative for the use on
the equine AI.