Boar semen freezing and sperm sexing in a specific disease-free nucleus herd

Abstract

The factors influencing of boar semen production were evaluated a total of 19,966 ejaculates from 517 PRRS free boars (2 purebred; 164D, 31P) and (2 crossbred; 268LY, 54PD). Semen parameters; volume (ml), sperm concentration (x106 sperm/ml) and total number of sperm per ejaculate (x109 sperm/ejaculate) were evaluated in relation to seasons as summer (Mar-Jun), rainy (Jul-Oct) and winter (Nov-Feb) and breed differences. Seasoning effected, the total number of sperm per ejaculate in winter was higher than a period in rainy season (Aug-Oct) (P<0.05) while the concentration in early winter (Nov and Dec) was lower than summer and a month in rainy (Jul) (P<0.05). Affect from breed, the total sperm production of LY was higher than D and P (P=0.03). Fertility data using D semen (TB and BA) tended to increase during Mar-Jun and Oct-Dec but the lowest TB and BA in Oct (P< 0.05). It can be concluded that the sperm production was influenced both of breeds and seasons what was found in PRRS free boars kept in EVAP in Thailand. Boar semen freezability and in vivo fertility EXP1 was to evaluate the sperm motility of FT boar semen including motility, PMI and motility pattern by using CASA after thawing (T0) and at 60 min (T60) after incubation at 38°C. Forty-one ejaculates from 14L, 12Y and 15D boars were freeze-thawed and evaluated. Motion parameters including VCL(μm/s), VSL(μm/s), VAP(μm/s), LIN(%), ALH (μm) and total motility (MS-CASA;%) were measured. The results revealed that total motility and VSL of T0 differed (P<0.05) among breeds. Some motion characteristics of FT boar semen i.e., VSL, VAP, VCL and ALH significantly decreased an hour after post-thawing (P<0.05). However, there was no significant difference in MS-CASA, and LIN between T0 and T60 groups. EXP2 was to study the freezability and influence of 50%HSP as thawing medium and fertility data in sows after IUI with HFT semen in field. Semen from one hundred-fifteen boars including purebred (4B, 26D) and crossbred (53LY, 29PD) were classified as 3 groups by age and freeze-thawed with and without presenting 50% HSP then evaluated by CASA for classification as good or poor freezability. For artificial insemination, eighty-six sows were inseminated as 43 control sows (fresh semen) and 43 treatment sows (HFT semen). All sows in treatment group were induced estrus by PG600®. The percentage of subjective motility of crossbred was significantly different higher than purebred (P<0.05). In addition, post thawed motility in boar, age >2 yr, in LY and PD was higher significant different (P<0.05) while D was lower significant different (P<0.05). For thawing, of ten were classified as good freezability from fifty-three boars showed significant difference in motility, MS-CASA, VAP, VSL, LIN and viability (P<0.05) between BTS and 50%HSP groups. However, the beneficial effected of 50%HSP did not present in our study. For fertility data, there were no significant difference between control and treatment groups in FR, TB and BA (P>0.05). In summary, boar individuality with good freezability should be tested for achievement fertility data in field. Boar semen sexing using percoll gradient and in vivo testing after insemination The objectives aimed to study the possibility to use discontinuous gradient centrifugation to sex boar spermatozoa and test by in vivo fertilization. To evaluate the efficiency of percoll-gradient centrifugation for sperm sexing, semen samples from fifteen ejaculates of five boars were studied (three replications). Eight layers of percoll-gradient concentration was used to separate semen as 90, 80, 75, 60, 55, 45, 30 and 20%, respectively. Fresh semen was placed on top and then centrifuged. Before (fresh semen) and after centrifugation (upper part; concentration 45% and 55% and lower part; concentration 80% and 90%) were collected into each aliquot; one, unprocessed initially fresh semen/ the other two aliquots, upper and lower part, respectively. All samples were extracted DNA then processed by modified quantitative PCR to calculate the percentage of X- and Y- bearing spermatozoa with standard curve. Two sets of primers were designed on specific AMELX and Y-chromosome (SRY) genes with SYBR green. The percentage of difference in each X- and Y- spermatozoa population was compared to initial fresh semen. In vivo testing, two boars with presenting the normal fertility were included in this study. The control group (unsex semen) was routinely processed while the treatment group (sexed semen) was processed by discontinuous percoll gradient centrifugation. The seventy-eight wean sows were included in this experiment. Seventy-eight sows were inseminated by IUI as control (N=39) with unsex semen and treatment groups (N=39) with the lower part of semen identified as X dominance. The 21-day CR, FR, TB and sex ratio of total born (female:male) were collected to compare as P<0.05. The result of the difference of percentage of X- and Y- bearing spermatozoa population showed significant difference (P<0.05) in lower part to which was compared each individual fresh semen. Unfortunately, the result did not relate to the significant difference all of the parameters (P>0.05) for the fertility data, however, the sex ratio of total born (female:male) in treatment group seems higher than control. In conclusion, the significant difference in percentage of X- spermatozoa populations by percoll gradient centrifugation can be enhanced by discontinuous percoll gradient centrifugation. Nevertheless, the effect of sex ratio from in vivo fertilization did not relate closely to the insemination with the shifting of the difference of X- or Y- spermatozoa populations. The results of all studies indicated that breeds and seasons could effect on semen production of boar kept in EVAP. In addition, the classification of boar as freezabiltiy to preserve their genetics as frozen semen form should be done to be acceptable fertility data. Percoll-gradient centrifugation can slightly shift the percentage of sex of sperm population, however, it did not significantly relate to sex ratio of TB. Finally, the application of reproductive technology should be simplified and adapted with easy and economical concepts but high efficiency for commercial pig production in field.