Abstract
Male fertility involves the capacity to obtain viable pregnancy and offspring after insemination. Currently, the most common way to measure bull fertility is through non-return rates (NRR) calculated after insemination of many females. However, this method is time-consuming and expensive. A number of biochemical molecules in sperm have been proposed as potential predictors of male fertility, e.g. platelet-activating factor (PAF). Platelet-activating factor (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphorylcholine) is a ubiquitous phospholipid that is implicated in the mediation of a wide variety of reproductive processes. The mechanism of PAF’s action is a receptor-mediated event reported to affect intracellular calcium levels. Bull sperm contain PAF and its content has a positive relationship with motility. While the PAF-receptor has been reported in other species, it has not been demonstrated in bull sperm. Therefore, our objectives were to determine: 1. the relationship between PAF content in bull sperm and Estimated Relative Conception Rates (ERCR, a 3-year rolling average of NRR); and 2. the presence of the PAF-receptor in bull sperm. Sperm PAF content for bulls (n=8) with different ERCR was determined by radioimmunoassay. PAF-receptor expression was determined as follows: total RNA was purified by acid phenol extraction and ethanol precipitation. Complementary DNAs were synthesized by reverse transcriptase with dNTPs and random primers at 37°C, 60min; followed by 65°C, 5min. Reverse transcription (cDNA) products were amplified with Taq polymerase, dNTP, and PAF receptor primer pair (upper, 5′-AATCCAGTCACCCTGGTTGTAG-3′; lower, 5′-TGGACTCAGAGTTCCGATACAC-3′) at 94°C, 1min; 55°C, 1min; 72°C, 1min for 35 cycles followed by 72°C, 7min. RT-PCR products were analyzed by 2% agarose gel electrophoresis. PAF-receptor protein was determined as follows: PBS-washed bull sperm was exposed to human PAF-receptor antibody at 4°C for 3h, washed in PBS, then exposed to fluorescein isothiocyanate-conjugated anti-IgG for 90min at 37°C, and again washed in PBS. Specimens were examined by epifluorescence microscopy at 400×. PAF content in bull sperm ranged from 1.39ng/106 sperm cells to 13.68ng/106 sperm cells. There was a positive correlation (P<0.05) between PAF content and ERCR. Presence of PAF-receptors in bull sperm was confirmed by immunofluorescence. However, distribution of PAF-receptors in bull sperm was not uniform within or between specimens. A cDNA clone containing the coding region for PAF-receptor was isolated from bull sperm using a reverse transcription-polymerase chain reaction protocol. There is a positive correlation (R=0.40; P<0.05) between PAF content in sperm and in vivo fertility of individual bulls as determined by NRR. Molecular and immunofluorescence data confirm the presence of PAF-receptor (mRNA and protein) in bull sperm. Additional studies are warranted to elucidate the mechanism of PAF’s action in sperm. Early selection for fertility in bulls represents a potentially valuable application to enhance efficiency in cattle breeding.
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