Research articleEffect of duration of storage at ambient temperature on fertilizing ability and mucus penetration ability of fresh bovine sperm
Introduction
Artificial insemination is the method of choice for dissemination of genetic improvement. The use of fresh semen for AI enables a 10-fold reduction of the insemination dose compared with frozen-thawed semen without a reduction in pregnancy rates [1], [2] thus allowing the more widespread use of high genetic merit sires. Recent world statistics for AI in cattle stand at 232 million doses of frozen and 11.6 million of fresh semen, the latter restricted primarily to New Zealand with smaller amounts used elsewhere [3]. The process of freezing and thawing irreversibly affects a significant proportion of sperm with typical estimates of 50% survival postthawing.
The potential genetic contribution of a sire is determined by the number of progeny he leaves and his genetic superiority. In cattle, the number of progeny is determined by (1) the total sperm output of the bull, (2) the number of sperm used per insemination, and (3) the percentage of cows calving to a single insemination. The average sperm production of a bull ranges from approximately 4 to 12 × 109 sperm per ejaculate. Typically, when using liquid semen, a minimum number of 2.5 × 106 total sperm per dose is required. Consequently, each ejaculate could provide up to 4800 doses. However, when using frozen semen, approximately 20 million total sperm per dose are required, amounting to a maximum of about 600 doses of frozen semen per ejaculate. On average, the number of semen doses possible for a single sire in a calendar year ranges between 100 000 and 150 000 if used exclusively as frozen semen. However, the seasonal mating patterns in some countries such as New Zealand, Australia, and Ireland, allows distribution of semen in liquid form. The advantages of using fresh semen include the ability to use low sperm numbers per insemination, high sire utilization, inexpensive storage, and ease of use in the field. However, this form of storage lacks the flexibility of frozen semen due to its finite shelf life [2], [4].
Storage of bovine semen in the fresh and frozen state has been comprehensively reviewed [2]. Bovine spermatozoa stored at ambient temperature in Caprogen diluent show a slow decrease in motility over an extended period about 3 to 4 wk, but the associated decrease in fertility is significantly higher [1]. Fertility, as measured by nonreturn rate, is maintained for the first 3 to 5 d after dilution, followed by a rapid decrease in non return rate until Day 10 of storage [1]. The physiological reasons for this rapid decline in fertility of spermatozoa stored at ambient temperature is presumed to be due to extracellular oxidative stress, effects of seminal plasma, and endogenous free radical production [1].
Although the use of fresh semen in the Irish dairy AI industry only accounts for 5% of total AI usage, this may peak to over 25% during the spring breeding season due to the increased demand for Irish proven sires of high genetic merit. With such sires, this can result in insufficient semen being available during periods of highest demand. Currently, in Ireland, fresh semen is only used for 2 d postcollection due to the perceived drop in pregnancy rates subsequently. The ability to use fresh semen at low dose rates over a period of 3 to 4 d would significantly improve the number of inseminations per ejaculate per bull.
The objective of this study was to examine the effect of storage of fresh semen for up to 7 d at ambient temperature on fertilization and embryo development in vitro, and on the ability of sperm to penetrate artificial mucus in vitro.
Section snippets
Semen collection and processing
Semen from Holstein Friesian bulls standing at an AI center was used throughout (National Cattle Breeding Centre, Enfield, Ireland). Semen was collected by artificial vagina and split for conventional freezing or fresh processing. Fresh semen was collected and diluted to 20 × 106 sperm/mL in Caprogen diluent, stored at room temperature and used to fertilize in vitro matured (IVM) oocytes at 5 to 8 h post semen collection (the time taken to collect, process and transport the semen to the IVF
Experiment 1: effect of duration of storage of fresh bovine semen on embryo development in vitro following IVF
There was no bull effect (P > 0.05) or duration of storage effect (P > 0.05) on oocyte cleavage rate or blastocyst development after IVF. Irrespective of sire, storage of fresh semen at ambient temperature for up to 4 d postcollection had no effect on cleavage rate or blastocyst development after IVF with Percoll-separated sperm (Fig. 1). In addition, blastocyst quality, as assessed by the proportion of blastocysts hatching from the zona, was not affected by semen storage. Similarly, when IVF
Discussion
The main findings of this study are (1) fresh bovine sperm stored at room temperature are capable of fertilizing oocytes in vitro for at least 7 d without any reduction in development, and (2) storage of fresh sperm at room temperature for longer than 2 d results in a significant reduction in their ability to penetrate artificial mucus.
In contrast to natural mating in cattle, during which approximately 10 × 109 sperm are deposited in the anterior vagina, AI bypasses the cervical barrier,
Acknowledgments
The authors thank the staff of the National Cattle Breeding Centre, Enfield, County Meath, Ireland, for provision of semen and to the staff of Kildare Chilling, County Kildare, Ireland, and Kepak, Clonee, County Meath, Ireland, for allowing access to bovine ovaries. J.I. Marti was supported by a grant from Programa Europa CAI-CONSI+D. A. Al Naib was funded by the Syrian University of Aleppo.
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