• Journal of Embryo Transfer
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• v.24 no.3
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• pp.225-230
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• 2009

Sex-sorting of sperm is an assisted reproductive technology (ART) used by the livestock industry for the mass production of animals of a desired sex. The standard method for sorting sperm is the detection of DNA content differences between X and Y chromosome-bearing sperm by flow cytometry. However, this method has variable efficiency and therefore requires verification by a second method. We have developed a sex determination method based on quantitative real-time polymerase chain reaction (qPCR) of the porcine amelogenin (AMEL) gene. The AMEL gene is present on both the X and the Y chromosome, but the length and sequence of its noncoding regions differ between the X and Y chromosomes. By measuring the threshold cycle (Ct) of qPCR, we were able to calculate the relative frequency of X chromosome. Two sets of AMEL primers were used in these studies. One set (AME) targeted AMEL gene sequences present in both X and Y chromosome, but produced PCR products of different lengths for each chromosome. The other set (AXR) bound to AMEL gene sequences present on the X chromosome but absent esholthe Y-chromosome. Relative product levels were calculated by normalizing the AXR fluorescence to the AME fluorescence. The AMEL method accurately predicted the sex ratios of boar sperm, demonstrating that it has potential value as a sex determination method.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.2
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• pp.91-98
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• 2021

The objective of this study was to establish a selection process for high quality sperm in bovine semen using sperm separation by magnetic activation (MACS). For this, semen from 21 Nellore bulls was collected using an artificial vagina. To guarantee the presence of pathologies in the ejaculate, animals previously declassified in four consecutive spermiogram were used. Semen was analyzed in five statuses: (1) fresh semen (fresh); (2) density gradient centrifugation (DGC), percoll column; (3) non-apoptotic fraction after separation by MACS (MAC); (4) apoptotic fraction from the separation (MACPOOR); and (5) MAC followed by DGC (MACDGC). Using a computerized analysis system (CASA), motility was measured. The sperm morphology was evaluated by phase contrast, and the supravital test was completed with eosin/nigrosin staining. For DGC, 20 × 10⁶ cells were used in a gradient of 90% and 45% percoll. MACS used 10 × 10⁶ cells with 20 μL of nanoparticles attached to annexin V, and filtered through the MiniMACS magnetic separation column. Membrane integrity was assessed with SYBR-14/IP and mitochondrial potential with JC-1 by flow cytometry. Processing sperm by MACDGC, was more effective in obtaining samples with high quality sperm, verified by the total of abnormalities in the samples: 35.04 ± 2.29%, 21.50 ± 1.47%, 17.30 ± 1.10%, 30.68 ± 1.94% and 10.50 ± 1.46%, respectively for fresh, DGC, MAC, MACPOOR, and MACDGC. The subpopulation of non-apoptotic sperm had a high number of live cells (82.65%), membrane integrity (56.60%) and mitochondrial potential (83.98%) (p < 0.05). These findings suggest that this nanotechnological method, that uses nanoparticles, is efficient in the production of high-quality semen samples for assisted reproduction procedures in cattle.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.121-128
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• 2015

The objective of this study was to evaluate the efficiency of sperm cryosurvival in boar sperm separated by Percoll containing antioxidant enzymes. The boar semen was collected into a pre-warmed ($37^{\circ}C$) thermos bottle by gloved-hand method and was separated by 65% Percoll with superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) before freezing. The frozen sperm was thawed at $38.5^{\circ}C$ for 45 sec in water-bath for sperm characteristic analysis. The sperm were estimated with SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction, Rhodamine123/PI double staining for mitochondrial integrity and were analyzed using flow cytometry. In results, sperm viability, acrosome reaction and mitochondrial integrity were improved in separated sperm groups compared with unseparated sperm by Percoll (UP) group. Especially, viability was significantly higher in sperm separated by Percoll containing 400 IU CAT group compared with other groups (P<0.05). And acrosome reaction was decreased in sperm separated by Percoll with 300 IU SOD, 400 IU CAT and 0.5 mM GSH groups compared with other groups, however, there were no significantly difference mitochondrial integrity among sperm separated by Percoll with antioxidant enzymes. In conclusion, we suggest that use of Percoll containing antioxidant enzymes for sperm separation will be beneficial for sperm cryopreservation in pigs.

• Journal of Embryo Transfer
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• v.23 no.4
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• pp.275-281
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• 2008

The ability to preselect the sex of piglets is advantageous in the pig industry. The objective of this study was to examine the feasibility of using intracytoplasmic sperm injection (ICSI) with sorted spermatozoa to produce piglets with a preselected sex. Pig embryos were produced by ICSI of frozen X- and Y-sperm that had been separated by flow cytometry. The developmental competence of the embryos was investigated in vitro and in vivo. The populations of X- and Y-spermatozoa were 52.7% and 47.3%, respectively in our samples. The in vitro development of ICSI embryos was enhanced by longer of in vitro maturation of oocytes ($44{\sim}48\;h$ vs. $40{\sim}43\;h$). Their cleavage ($65{\sim}70%$) and blastocyst formation ($9{\sim}12%$) rates were not significantly different between male and female ICSI embryos, or between sorted and unsorted sperm-derived embryos. One pregnancy was established in a recipient that was transferred with 110 female ICSI embryos, but the pregnancy was terminated on Day 89 of gestation. Our results suggest that the separation X- and Y-spermatozoa by flow cytometric sorting can be a useful tool in combination with ICSI for the production of pig embryos and piglets of preselected sex.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.181-186
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• 2011

Spermatozoa sorted by flow cytometry have been successfully used to produce offspring in domestic animals and are commercially available for cattle. Also sheath fluid is the important environment for viability of sex-sorted sperm in flow cytometry. The aim of this study was to investigate whether or not HEPES (N-2-hydroxyethylpiperazine-N'-2-Ethanesulfonic acid) has any effect on the viability in sex-sorted Hanwoo (Korean native cattle) sperm. In this study, the semen was collected from Hanwoo of Hoengseong Livestock Cooperation by artificial vagina method then pooled and subjected to cryopreservation in straws. Sperm were cultured for 0, 30, 60 and 120 min with 0, 2.5, 5, 7.5 and 10 mM of HEPES added to the sheath fluid and incubated at 4, 20 and 38$^{\circ}C$, respectively. For the cytometric analysis the frozen-thawed semen was extended with 5 mM HEPES extender to final concentration ($2{\times}10^7$ spermatozoa) at 4, 20 and 37$^{\circ}C$. Sperm viability was assessed with SYBR-14 and propidium iodide (PI) staining. This study shows that the viability of sperm was decreased with prolongation of incubation time in all of test. But the viability of sperm which were treated with 38$^{\circ}C$ was gently decreased than that of treated with other temperature. The viability of the control was sharply decreased (p<0.05) than all of the HEPES treatment group at 60 to 120 min in 38$^{\circ}C$. X-sexed sperm was more sensitive than Y-sexed sperm to temperature during f10w cytometry (p<0.05). In conclusion, the results of this study suggest that the sheath fluid with 5 mM HEPES has effect on maintenance of viability after sperm sexing at 37$^{\circ}C$ in Hanwoo.

• Journal of Embryo Transfer
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• v.29 no.3
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• pp.283-287
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• 2014

Sexed semen is commonly used for the production of calves of the desired gender. Gender selection is important in animal production industries. For example, female cattle are required for the dairy industry while males are preferred in the beef cattle industry. The present study was to assess the in vivo embryo production efficiency using the semen separated according to sex during superovulation in Hanwoo. Seventy Hanwoo donor cows were flushed on day 7 of estrus cycle with same FSH and artificial insemination by the same technicians. Embryos were recovered on 7 days after the third insemination by flushing the uterus with embryo collection medium. KPN semen straws used artificial insemination contained 20 million sperm (total number 60 million per donor). Sex-sorted semen straws contained 4 million sperm (total number 12 million per donor). The results obtained were as follows: No differences were observed in the efficiency of superovulation rates on KPN semen 87%, and sexed semen 100%, respectively. The mean numbers of total embryos are each $12.58{\pm}8.31$ and $13.25{\pm}7.86$. The mean numbers of transferable embryos, sexed semen were significantly lower than KPN semen ($3.75{\pm}1.98$ vs. $8.23{\pm}6.07$, P<0.05). The rates of unfertilized embryos from superovulation using sexed semen were significantly higher than KPN semen (50% vs. 15%, P<0.05). The rate of degenerated 2-cell embryos from sexed and KPN semen was 60.87% and 11.11%, respectively (p<0.05). In conclusion, these results indicate that superovulation using sexed semen was useful, but efficient embryo production was important to reducing the damage caused by the Flowcytometer-based sperm sorting procedure.

• Korean Journal of Poultry Science
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• v.39 no.4
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• pp.291-295
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• 2012

The use of methylacetamide (MA) as a cryoprotective agent for freezing Korean Native Black rooster Ogye semen was examined with artificial insemination. The diluted Ogye semen with HS-1 was subjected for 2 step dilution method of cryopreservation in which the final concentration of MA was adjusted to 7.5%. The sperm viability after thawing was reduced from $95.17{\pm}0.93%$ to $55.93{\pm}1.38%$ which was confirmed by live-death analysis based on Fluorescence-Activated Cell Sorting (FACS). The rates of fertilized eggs with fresh or frozen-thawed semen were reduced from $94.98{\pm}3.93%$ to $66.36{\pm}8.43%$ at day 7 with significant difference. However, the hatching rates of experiments at day 21 did not shown difference between $92.64{\pm}2.33%$ and $90.45{\pm}8.05%$ (P<0.05). With these results, the utilization of MA for freezing of Ogye spermatozoa could affect on viability of frozen-thawed semen but not on the fertility of lain eggs and hatchability of fertilized eggs and also provide possible tools of freezing for poultry genetic resource conservation.