Sperm sexing in Nili-Ravi buffalo through modified swim up: Validation using SYBR ® green real-time PCR

Asma-Ul-Husna,; Awan, Muhammad Amjad; Abid, Muhammad; Sultana, Tasawar; Shahzad, Qaisar; Ansari, Muhammad Tayyab; Rakha, Bushra Allah; Naqvi, Syed Muhammad Saqlan; Akhter, Shamim

doi:10.1016/j.anireprosci.2017.04.011

Cited by 29 publications

(22 citation statements)

References 53 publications

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“…Madrid-Bury et al (2003) isolated the X/Y spermatozoa from Holstein bulls using an upstream method (addition of heparin), and the percentage of Y spermatozoa in the upper sperm decreased after 150 min of incubation. Asma Ul et al (2017) also isolated X/Y spermatozoa from buffaloes using a modified upstream method. He discovered that the percentage of Y sperm cells accounted for four times more than the X sperm (Asma Ul et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

“…Asma Ul et al (2017) also isolated X/Y spermatozoa from buffaloes using a modified upstream method. He discovered that the percentage of Y sperm cells accounted for four times more than the X sperm (Asma Ul et al, 2017). In addition, the researchers studied the relationship between pH and the sex ratio of offspring by measuring the pH of the reproductive tract during insemination or changing the pH of the semen incubation solution.…”

Section: Discussionmentioning

confidence: 99%

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Effects of Diluent pH on Enrichment and Performance of Dairy Goat X/Y Sperm

Huang

et al. 2021

Front. Cell Dev. Biol.

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In this paper, on the basis of the differences in the hydrogen ion concentration (pH) of the diluent dairy goat semen on X/Y sperm motility, an X/Y sperm enrichment study was conducted to establish a simple and effective method for gender control in dairy goats. Dairy goat semen was diluted using different pH dilutions and was incubated. Then, the X/Y sperm ratio in the isolated upper sperm was determined using the double TaqMan qPCR method. The internal pH change pattern of sperm cells at different pH dilutions was measured using BCECF-AM probe, and the functional parameters of the isolated sperm were tested with the corresponding kit. Next, an in vitro fertilization test was conducted using isolated spermatozoa and oocytes to determine their fertilization rates, the percentages of female embryos, and the expression of genes related to developing potentially fertilized embryos. Results showed that the percentages of the X sperm cells in the upper sperm layer were 67.24% ± 2.61% at sperm dilution pH of 6.2 and 30.45% ± 1.03% at sperm dilution pH of 7.4, which was significantly different from 52.35% ± 1.72% of the control group (pH 6.8) (P < 0.01). Results also showed that there is a relationship between the external pHo and internal pHi of sperm cells. Furthermore, the percentages of female embryos after the in vitro fertilization of the isolated upper sperm with mature oocytes at pH 6.2 and 7.4 were 66.67% ± 0.05 and 29.73% ± 0.04%, respectively, compared with 48.57% ± 0.02% in the control group (pH 6.8). Highly significant differences occurred between groups (P < 0.01). Additionally, no significant difference was observed during the expression of genes related to embryonic development between the blastocysts formed from sperm isolated by changing the pH of the diluent and the control sperm (P > 0.05). Therefore, this study successfully established a simple and effective method for enriched X/Y sperms from dairy goats, which is important for regulating the desired sex progeny during dairy goat breeding and for guiding dairy goat production.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Effects of Diluent pH on Enrichment and Performance of Dairy Goat X/Y Sperm

Huang

et al. 2021

Front. Cell Dev. Biol.

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“…The Percoll density gradient centrifugation is based on differences in X-and Y-sperm DNA content, allowing for cell separation. After centrifugation, X-bearing sperm cells settle at the lower layer, while the Y-bearing sperm cells remain at the upper level (supernatant) [2]. Sexing sperm cells using the swim-up method are based on differences in motility speed of swimming out of the pellets to the surface of the media.…”

Section: Introductionmentioning

confidence: 99%

The enriched Y-bearing sperm combined with delayed fixed-time artificial insemination for obtaining male Simmental crossbred offspring

et al. 2022

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Background and Aim: The production of male calf beef cattle is an agricultural innovation needed to increase the farm's productivity as a provider of meat sources. This study aimed to determine the sex ratio of the offspring of cows inseminated with Y-bearing sperm enriched by Percoll density gradient centrifugation and swim up, combined with delayed fixed-time artificial insemination (FTAI). Materials and Methods: Ejaculates of Simmental bulls were divided into four equal portions and grouped as T0 (control, non-sexed semen), T1 and T2 were sexed semen using Percoll density gradient centrifugation three and five levels, respectively, and T3 was sexed semen using swim-up. After the sex was sorted, the semen was diluted in a tris egg yolk extender, packaged in French mini-straws containing 50 million live sperm cells, and frozen. Pre-sexed, post-sexed, and post-thawed spermatozoa were evaluated based on progressive motility, viability, intact plasma membrane, and abnormality. The post-thawed semen of T0 was artificially inseminated to recipient cows at 12 h after onset of estrus (not delayed FTAI). Meanwhile, the delayed FTAI was conducted 18 20 h after onset of estrus using the T0, the best of T1 and T2, and the T3 post-thawed semen. Results: The Percoll density gradient centrifugation reduced motility, viability, and intact plasma membrane but increased sperm abnormalities. Meanwhile, the swim up process increased motility, viability, and intact plasma membrane of sperm cells but decreased sperm abnormalities. Post-thawed semen decreased motility, viability, and intact plasma membrane of sperm cells but increased sperm abnormalities. The sex ratio of the Simmental crossbred offspring was 96.08% and 100% in T1 and T3, respectively, compared to 48.25% and 67.39% in T0 not delayed and delayed FTAI, respectively. Conclusion: The Percoll density gradient centrifugation and swim-up methods are prospective for obtaining male offspring.

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“…Thus, it is not a surprise that the use of sex-sorted sperm in bovine assisted reproduction has steadily increased in the last years [4,5]. Although alternative methodologies have been described [6][7][8], the separation of X-and Y-bearing spermatozoa by means of flow cytometry after Hoechst 33342 labeling is still the technique of choice applied in most sorting facilities, mainly due to its high accuracy, repeatability and suitability for commercial application [9]. Nevertheless, several research groups had already reported that inseminating dairy heifers with a dose of 1 to 2 million frozen-thawed Xbearing sperm resulted in conception rates not higher than 70-90% of these achieved with unsorted sperm (henceforward mentioned as "conventional" in the text; [10][11][12][13][14][15]).…”

Section: Introductionmentioning

confidence: 99%

The micro-RNA content of unsorted cryopreserved bovine sperm and its relation to the fertility of sperm after sex-sorting

et al. 2021

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Background The use of sex-sorted sperm in cattle assisted reproduction is constantly increasing. However, sperm fertility can substantially differ between unsorted (conventional) and sex-sorted semen batches of the same sire. Sperm microRNAs (miRNA) have been suggested as promising biomarkers of bull fertility the last years. In this study, we hypothesized that the miRNA profile of cryopreserved conventional sperm is related to bull fertility after artificial insemination with X-bearing sperm. For this purpose, we analyzed the miRNA profile of 18 conventional sperm samples obtained from nine high- (HF) and nine low-fertility (LF) bulls that were contemporaneously used to produce conventional and sex-sorted semen batches. The annual 56-day non-return rate for each semen type (NRRconv and NRRss, respectively) was recorded for each bull. Results In total, 85 miRNAs were detected. MiR-34b-3p and miR-100-5p were the two most highly expressed miRNAs with their relative abundance reaching 30% in total. MiR-10a-5p and miR-9-5p were differentially expressed in LF and HF samples (false discovery rate < 10%). The expression levels of miR-9-5p, miR-34c, miR-423-5p, miR-449a, miR-5193-5p, miR-1246, miR-2483-5p, miR-92a, miR-21–5p were significantly correlated to NRRss but not to NRRconv. Based on robust regression analysis, miR-34c, miR-7859 and miR-342 showed the highest contribution to the prediction of NRRss. Conclusions A set of miRNAs detected in conventionally produced semen batches were linked to the fertilizing potential of bovine sperm after sex-sorting. These miRNAs should be further evaluated as potential biomarkers of a sire’s suitability for the production of sex-sorted sperm.

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Sperm sexing in Nili-Ravi buffalo through modified swim up: Validation using SYBR ® green real-time PCR

Cited by 29 publications

References 53 publications

Effects of Diluent pH on Enrichment and Performance of Dairy Goat X/Y Sperm

Effects of Diluent pH on Enrichment and Performance of Dairy Goat X/Y Sperm

The enriched Y-bearing sperm combined with delayed fixed-time artificial insemination for obtaining male Simmental crossbred offspring

The micro-RNA content of unsorted cryopreserved bovine sperm and its relation to the fertility of sperm after sex-sorting

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