Abstract
This study aimed to establish a protocol for in vitro embryo production using epididymal sperm (EP). Samples were obtained from ejaculated sperm (EJ) and the epididymis of 7 Gir bulls. First, the effect of heparin (+) on the viability, longevity (Experiment 1) and fertilization rates (Experiment 2) of the EP was evaluated. In experiment 2, a pool of EP and EJ sperm (n = 7) was coincubated with cumulus-oocyte complexes (COCs) for 0, 3, 6, 12 and 18 h, and the fertilization rate (FR) was evaluated. A third experiment was performed to test sperm treatments for IVP using the Percoll (P) or PureSperm (PS) gradients or a spTALP wash for sperm selection. Cleavage, blastocyst rate (BR) and embryo sex were evaluated. In experiment 4, embryos were produced using 6, 12, and 18 h of sperm-oocyte coincubation. The cleavage, BR, and total number and percentage of apoptotic cells were determined. Heparin affected EP viability, longevity and FR. After 6 h, 82% of the oocytes were fertilized in the EP+ group, a higher value (P<0.05) than that in the EJ (19%) and EP- (42%) groups. At 12 and 18 h, FR remained higher in the EP+ group, and a gradual increase in polyspermy was observed. The use of a P or PS gradient yielded a similar BR on D7 (54% and 52%), which was higher than the rate obtained using the washing method (37%). The embryos produced by EP and selected in a P or PS gradient resulted in a sex deviation in favor of male embryos (P>0.05). No differences (P>0.05) were observed among the groups that were coincubated for 6, 12 and 18 h with respect to embryo production, kinetics of development, total cell number and percentage of apoptotic cells. In conclusion, IVF time can be reduced to 6 h without affecting embryo production and quality. In addition, EP sperm selection can be performed by either a PS or P gradient.
Highlights
Genetic material from animals of economic interest, wild animals or endangered species may be lost at any time by unexpected death or by acquired reproductive failure
A decrease in total and progressive motility was observed for the ejaculated sperm (EJ)+ and epididymal sperm (EP)+ groups after 3 h of incubation, while in the group EP, this decrease only occurred after 6 h for total motility and after 12 h for progressive motility
The results obtained when these sperm cells are used in assisted-reproduction techniques (ARTs) are inconsistent, which is probably due to lack of knowledge regarding their physiology and due to treating them in a similar manner as for the EJ
Summary
Genetic material from animals of economic interest, wild animals or endangered species may be lost at any time by unexpected death or by acquired reproductive failure. In most of these cases, there is a loss of genetic material, as well as an economic loss. The recovery and cryopreservation of epididymal sperm (EP) is one of these alternatives, because this process allows the preservation of male gametes and the maintenance of germplasm banks [1, 2] These spermatozoa can be used in artificial insemination (AI) or in vitro embryo production (IVP), either by intracytoplasmic injection (ICSI) or by in vitro fertilization (IVF) [3, 4]
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