Economic Aspects and Evaluation of the Viability Period of Immunosexed and Diluted Ram Sperm in Powdered Coconut Water Conservation Extender (ACP-102c)

Luiz Carlos Pinheiro Maia, Bruna Farias Brito, Bárbara Mara Bandeira Santos, Leonardo Alves Rodrigues Cabral, Cristiane Clemente de Mello Salgueiro, Marcos Fernando de Resende Matta, José Ferreira Nunes


Background: Sperm sexing is increasing in use because pre-determining the sex of the calf allows greater profitability and promotes significant gains in the productive systems that utilize the technique. Deployment of a low-cost and practical preservation methodology may further favor the cost-benefit ratio. Flow cytometry, the most commonly used sexing technique, has high costs and is very restricted. As an alternative, immunosexing has been studied, which uses sex-specific monoclonal antibodies. Thus, the objective of this study was to evaluate the immunosexing technique in conjunction with cryopreservation in ACP-102c and examine its economic aspects with regard to ram semen.

Materials, Methods & Results: Ejaculates from two ram individuals were collected with the aid of an artificial vagina, evaluated, and submitted to the immunosexing protocol, according to the manufacturer's recommendations, using the Monoclonal Antibody Kit specific for mammalian sperm with “Y” chromosomes (HY; HY Biotechnology, Rio de Janeiro-RJ, Brazil). After sexing, the supernatant was resuspended in the cryopreservation diluent: ACP (ACP-102c + 20% egg yolk + 7% glycerol), packaged in 0.25 mL straws, refrigerated to 4°C, stabilized for 30 min, frozen in liquid nitrogen vapors (-60°C) for 15 min, immersed in liquid nitrogen, and stored in cryogenic cylinders. The samples were thawed and evaluated for sperm kinetics both by using computerized semen analysis with SCA® software (Sperm Class Analyzer version 5.0) and subjectively comparing specimens from the two animals using conventional microscopy (40x). Plasma membrane integrity (IMP) and sperm cell morphology were evaluated by the smear staining technique using eosin-nigrosine dye, and the percentages of healthy and normal spermatozoa were determined. A bibliographic survey and a market study of similar products and technologies were carried out to provide an economic viability metanalysis of the bioproduct (ACP-102c) and bioprocess (immunosex). The data were analyzed using R-project©, and comparisons made between animals and between thawing periods using T test. There were no statistically significant differences between animals and between periods (P > 0.05), except for the normal sperm parameter, in which animal A1 had the lowest percentage (P < 0.05). As for the cost-benefit ratio, flow cytometry as a technique is more laborious and expensive, while immunosexing associated with cryopreservation in ACP-102c diluent has proven more practical, with regards to both sperm sexing techniques and diluents for sperm conservation.

Discussion: In general, the quality of cryopreserved sexed semen was lower than that of non-sexed semen; however, in this study, both in the comparison between animals and between evaluation periods, similar values of motility, viability, and sperm morphology were obtained for sexed and several non-sexed cryopreserved semen samples, demonstrating that immunosexing did not severely affect the sperm structure, and that the ACP-102c conservation medium was efficient at maintaining the plasma membrane of these sperm. In the evaluation of economic aspects, it was observed that immunosexing, associated with cryopreservation in ACP-102c diluent, proved to be the most practical technique, requiring only conventional equipment, and allowing a greater field of application, since the immunosexing semen can be used for primiparous and multiparous females. Thus, it was concluded that immunosexing associated with cryopreservation in an ACP-102c diluent was more cost-effective, more practical, and had significantly improved sperm quality results after sexing.

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