In vitro evaluation of the quality and fertilizing capacity of boar semen frozen in 0.25 ml straws.

Department of Animal Pathology (Animal Health), University of Leon, Leon, Spain.
Reproduction in Domestic Animals (Impact Factor: 1.39). 05/2006; 41(2):153-61. DOI:10.1111/j.1439-0531.2006.00659.x
Source: PubMed

ABSTRACT Twenty-two boar ejaculates were frozen in 0.25 ml straws using a controlled cooling rate, then evaluated in vitro in order to assess: (i) the extent to which a range of semen evaluation parameters accurately characterize sperm quality, (ii) the value of quality assessment in the characterization of long-term sperm survival and fertility and (iii) the suitability of the cryopreservation protocol used for yielding semen with good quality and fertilizing capacity. Motility with or without caffeine, plasma membrane integrity (PMI) evaluated with both propidium iodide (PI) and Hoechst 33258, and acrosome morphology were studied, the ejaculates being then classified into five quality groups. A thermoresistance test and a homologous in vitro fertilization test were applied to selected ejaculates of these groups. Caffeine-stimulated motility and PMI evaluated with PI provided better estimations of semen quality than the other tests of motility, PMI, or acrosome morphology, but this quality assessment could not reveal differences in fertilizing capacity or thermoresistance among ejaculates. Over 43% spermatozoa survived cryopreservation in 19 of the 22 ejaculates, with inter-boar and inter-ejaculate variability in the freezing success being observed. The fertilizing capacity, however, was seriously affected by the process regardless of the semen quality. It is concluded that caffeine-stimulated motility and PMI evaluated with PI give accurate information on sperm quality, but important aspects to the valuation of semen such as thermoresistance and fertilizing capacity are not revealed by this quality study. Moreover, the approach of selecting suitable protocols of cryopreservation does not appear to be sufficient for guaranteeing systematically good quality and fertilizing capacity in the frozen-thawed semen.

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    ABSTRACT: Cryopreservation induces extensive biophysical and biochemical changes in the sperm. In the present study, we used flow cytometry to assess the capacitation-like status of frozen-thawed boar spermatozoa and its relationship with intracellular calcium, assessment of membrane fluidity, modification of thiol groups in plasma membrane proteins, reactive oxygen species (ROS) levels, viability, acrosomal status, and mitochondrial activity. This experiment was performed to verify the effect of adding seminal plasma on post-thaw sperm functions. To determine these effects after cryopreservation, frozen-thawed semen from seven boars was examined after supplementation with different concentrations of pooled seminal plasma (0%, 10%, and 50%) at various times of incubation from 0 to 4 hours. Incubation caused a decrease in membrane integrity and an increase in acrosomal damage, with small changes in other parameters (P > 0.05). Although 10% seminal plasma showed few differences with 0% (ROS increase at 4 hours, P < 0.05), 50% seminal plasma caused important changes. Membrane fluidity increased considerably from the beginning of the experiment, and ROS and free thiols in the cell surface increased by 2 hours of incubation. By the end of the experiment, viability decreased and acrosomal damage increased in the 50% seminal plasma samples. The addition of 50% of seminal plasma seems to modify the physiology of thawed boar spermatozoa, possibly through membrane changes and ROS increase. Although some effects were detrimental, the stimulatory effect of 50% seminal plasma could favor the performance of post-thawed boar semen, as showed in the field (García JC, Domínguez JC, Peña FJ, Alegre B, Gonzalez R, Castro MJ, Habing GG, Kirkwood RN. Thawing boar semen in the presence of seminal plasma: effects on sperm quality and fertility. Anim Reprod Sci 2010;119:160-5).
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    ABSTRACT: Causes of poor fertility after insemination of frozen-thawed (FT) sperm include reduced sperm viability at thawing and a shorter longevity of surviving sperm in the female genital tract due to sub-lethal damage. The present studies examined the effect of incubating thawed boar sperm in seminal plasma (SP) on sperm membrane integrity (viability), and motility in vitro (experiment 1), and fertility in vivo (experiment 2). For experiment 1, FT sperm from five individual boars and a sperm pool from these boars were thawed and incubated for 4 h in media containing 0%, 10%, or 50% autologous seminal plasma (individual boars) or pooled seminal plasma (sperm pool). At approximately 10 min (0 h) and again at 1 h, 2 h, 3 h, and 4 h, sperm populations were examined for percentage sperm viability and percent sperm motility. Each variable progressively decreased during the incubation period. Incubation in 50% SP increased percentages of live sperm (P < 0.0001) and percent sperm motility (P < 0.01) at all time points compared to incubation in either 0% or 10% SP. For experiment 2, multiparous Large white x Landrace sows (n = 82) each received 900 IU eCG at weaning and 750 IU hCG 80 h later to control time of ovulation. Sows were assigned on the basis of parity to be inseminated with pooled semen with or without SP from the boars used in experiment 1. Sows received 3 x 10(9) live fresh-extended sperm (n = 30) or FT sperm thawed in 80 mL BTS extender (n = 26) or 3 x 10(9) live FT sperm thawed in 80 mL BTS containing 50% SP (FT-SP; n = 26). Sows were inseminated at 36 h, and 42 h after hCG injection. Compared to sows receiving fresh semen, the pregnancy rate of FT inseminated sows tended (P = 0.06) to be lower with the FT-SP group being intermediate. Farrowing rates were not different (83.3%, 69.2%, and 65.4% for fresh, FT, and FT-SP, respectively). Inseminations with FT sperm were associated with a reduction in litter size (P < 0.05), which was not evident in the FT-SP group. Taken together, these data confirm an adverse effect of inseminating FT sperm on sperm quality and sow fertility but suggest that thawing FT sperm in 50% SP may partially alleviate these adverse effects.
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