Improved fertility of flowcytometrically sex selected bull spermatozoa (2005)

The
 influence of flowcytometrical sorting and the effect of different
 modifications of extenders on the viability and fertilizing capacity of
 liquid and frozen/thawed bull spermatozoa were analysed in the present study.
 The study was divided in four experimental parts.
 The aims
 of the first study were to analyze effects of semen processing after sorting
 and subsequent freezing in liquid nitrogen on the quality and fertilizing
 capacity of bull spermatozoa. Semen was obtained from two fertility proven
 Holstein Friesian bulls. Quality of the frozen/thawed semen was evaluated by
 motility estimation, morphology analysis, membrane stability (6-CFDA/PI)
 test, capacitation test (FITC-PNA/PI staining with L-α-Lysophosphatidylcholine), and
 fertility assessment in an insemination trial on farm. A prolonged period
 after high dilution of sorted spermatozoa before freezing had a significantly
 negative effect on motility (P<0.001) and capacitation status (P<0.05).
 Positive effects on membrane stability were seen, when glycerol was added at 5°C shortly before straws were frozen, compared to a system where
 glycerol was added before the equilibration process at room temperature
 (P<0.001). Independently from sperm processing after sorting, sexed
 spermatozoa had significantly more damaged acrosomes and morphological
 abnormalities (P<0.001). In addition, sex sorted frozen/thawed spermatozoa
 (immediate centrifugation and glycerol addition at 5°C) and unsorted frozen/thawed semen samples were submitted to a
 thermo-resistance test at 37°C. Immediately after thawing no
 significant difference was seen in the percentage of motile spermatozoa
 between sorted and unsorted semen samples. However, after further incubation
 at 37°C, motility dropped significantly
 after 3 h (P<0.001) and 6 h (P<0.05). The fertilizing capacity of
 sex-sorted and unsorted spermatozoa from same ejaculates was tested in a
 field trial in heifers and cows with natural or synchronised oestrus cycle,
 respectively. In natural oestrus, more animals became pregnant after
 artificial insemination with unsorted than with sex-sorted spermatozoa (56.5
 % vs. 17.6 %; P<0.001). No significant differences were observed between
 unsorted and sex-sorted frozen/thawed semen samples after artificial
 insemination of the animals with synchronized estrus (36.4 % vs. 21.3 %).
 The goal
 of the second study was to investigate the effect of antioxidant
 supplementation on the quality of frozen/thawed sexed bull spermatozoa. Twelve
 ejaculates from two Holstein Friesian bulls were sorted according to the Beltsville
 Sperm Sexing Technology. Each ejaculate was divided into three parts and
 processed as unsorted control, according to a standard sorting protocol and
 in the presence of different antioxidants (S-AO). Cooling and freezing of the
 samples was performed equally for all three groups, except that antioxidants
 were added to TRIS-egg-yolk freezing extender for those semen samples that
 were already sorted in the presence of antioxidants. The semen quality in
 frozen/thawed samples was determined by morphology analysis immediately after
 thawing, motility estimation in a thermo-resistance test after 0, 6, 12 and
 24 h incubation at 37°C and FITC-PNA/PI staining after 0, 12
 and 24 h of incubation at 37°C. Assessment of motility showed
 significantly more (P<0.05) motile spermatozoa in S-AO samples as compared
 to unsorted frozen/thawed controls at 0, 6 and 24 h after thawing and
 compared to normally sorted samples at all times after thawing. Percentage of
 damaged acrosomes was significantly lower (P<0.05) in S-AO samples in
 comparison to unsorted control (20.8 ± 6.9 % and 30.3 ± 12.0 %, respectively)
 and the percentage of morphological abnormal spermatozoa was in this group
 significantly lower (P<0.05) in comparison to unsorted control and
 normally sorted samples (25.8 ± 5.2 %; 36.0 ± 12.5 % and 35.1 ± 7.4 %, respectively).
 Analysis of frozen/thawed spermatozoa with FITC/PI revealed no significant
 difference in membrane integrity at 0 and 12 hours after sorting, but after
 24 hours incubation the S-AO samples had significantly higher (P<0.001)
 percentage of spermatozoa with intact membranes in comparison to unsorted
 control and normally sorted semen (40.7 ± 6.3 %; 7.8 ± 4.7 % and 7.4 ± 4.6 % respectively).
 Percentage of acrosome reacted spermatozoa was significantly lower
 (P<0.05) in the S-AO samples in comparison to unsorted controls (14.1 ± 7.5 % vs. 25.9 ± 14.4 %;
 23.4 ± 5.4 % vs. 38.5 ± 16.7 % and 28.8 ± 6.3 % vs. 79.8 ± 4.1 %
 respectively) for 0, 12, 24 h after thawing and in
 comparison to normally sorted semen 24 hours after thawing (67.3 ± 10.0 %).
 This study shows for the first time the highly beneficial protective effect
 of antioxidative substances on the quality of sexed, frozen thawed bull
 semen.
 In the third study, modifications of a TRIS extender
 were used to balance major cells damages caused by the sorting process and
 liquid storing of the sorted spermatozoa. In order to prolong lifetime of
 sorted spermatozoa a special designed combination of antioxidants (AO) and
 bovine serum albumin (BSA) was used. The new extender increased the quality
 of sexed semen and no significant difference were seen in motility between
 unsorted controls and samples, sorted in the presence of AO and BSA. Similarly,
 membrane integrity as tested by FITC-PNA/PI did not differ significantly to
 controls. Acrosome integrity was significantly better in AO/BSA sorted group
 24 and 48 hours after incubation at 15°C
 (P<0.05) and also after 48 hours in the AO group without BSA (P<0.05)
 in comparison to unsorted control semen. There was no significant difference
 in pregnancy rates between unsorted control (66.7 %) and both sorted groups
 (AO/BSA (58.1 %) and AO (54.5 %) when semen was inseminated in
 non-synchronised heifers. Additionally, we could show for the first time that
 liquid sorted semen can be inseminated successfully within 72 hours after
 sorting.
 The aim
 of the fourth study was to analyse the effect of antioxidants and temporary
 chemical inhibition of sperm motility before and during flowcytometrical
 sorting on the quality and fertilizing capacity of sex sorted and
 frozen/thawed spermatozoa. The quality and fertilizing capacity of
 temporarily immobilized spermatozoa sorted in the presence of sodium fluoride
 (S-AO-NF) was compared with sorted semen using a standard protocol as
 developed in our laboratory (S-AO) and with unsorted control (C). Semen from
 two bulls was used and in total 283 first inseminations with 2 million live,
 frozen/thawed sex sorted and unsorted spermatozoa were performed on 197
 farms. After addition of sodium fluoride the motility of spermatozoa was
 completely inhibited and after sorting and centrifugation returned to a
 similar degree as before. Motility after thawing and incubation at 37°C did not differ between groups. After further incubation at 37°C for 12 h and 24 h motility was significant higher (P<0.001) in
 both sorted groups compared to unsorted semen (37.1 ± 4.9 %vs.
 33.1 ± 6.5 % vs. 7.5 ± 11.6 % and 22.9 ± 7.6 % vs. 21.3 ± 10.3 % vs. 0.0 ±
 0.0 % for S-AO-NF vs. S-AO vs. C for 12 and 24 hours respectively). The
 percentage of spermatozoa with acrosome abnormalities in frozen/thawed
 samples was significantly higher (P<0.05) in S-AO and C group when
 compared to S-AO-NF (23.0 ± 5.2 %, 22.3 ± 4.0 % and 14.7 ± 7.5 %
 respectively). The same difference was found for morphological sperm
 abnormalities (30.0 ± 6.7 %, 29.0 ± 5.8 % and 19.0 ± 8.1 % respectively). The
 percentage of acrosome reacted (AR) and viable spermatozoa was analysed by
 FITC-PNA/PI staining. The percentage of AR spermatozoa was significantly
 lower (P<0.001) in both groups of sorted semen when compared to unsorted
 control (10.6 ± 3.5 % vs. 6.7 ± 2.6 % vs. 19.1 ± 4.7 % for S-AO, S-AO-NF and
 C respectively). In addition, the percentage of viable spermatozoa was
 significantly higher (P<0.05) in the S-AO-NF group when compared to groups
 S-AO and C (72.1 ± 5.0 % vs. 56.9 ± 4.7 % vs. 61.4 ± 8.2 % respectively). Pregnancy
 rates after artificial insemination with the frozen/thawed semen were not
 significantly different between sorted spermatozoa and controls (72.7 % vs.
 73.3 % vs. 79.2 % for S-AO-NF, S-AO and C respectively). In
 conclusion, this study showed that reduction in quality and fertilizing
 capacity of sex sorted spermatozoa can be successfully prevented by
 modifications of extenders and handling of the semen before, during and after
 sperm sorting.