105 Increasing fertility of interspecific hybrid males using biotechnological approaches

2021 ◽  
Vol 33 (2) ◽  
pp. 159
Author(s):  
A. Vetokh ◽  
A. Tadzhieva ◽  
B. Iolchiev ◽  
N. Volkova ◽  
V. Bagirov

The results of AI depend on many factors, with the quality of semen being one of the most important. Not all male hybrids can meet the requirements for semen quality, because they often have reduced fertility following cryopreservation. Thus, it is necessary to improve semen processing before use in AI. The aim of the study was to evaluate the effectiveness of using the “swim-up” flotation method to improve sperm quality of hybrid males of the Ovis genus. Semen from interspecific hybrid rams (1/4 Argali×3/4 Romanov, n=15; 1/8 Argali×7/8 Romanov, n=15) was freshly obtained, frozen–thawed, and processed by the swim-up method. Evaluation of sperm motility was determined using computer-assisted semen analysis. Statistical analysis was performed using SPSS vs.15.0 (ANOVA and t-test; SPSS Inc.). Semen was collected during the breeding season (October–December) via artificial vagina. Assessment of acrosome integrity was determined using differential staining with a Diachem diff-quick kit (NPF ABRIS+). The degree of sperm DNA fragmentation was determined using the acridine-orange test. The sperm freezing/thawing cycle was accompanied by sperm damage and an increase in the proportion of immobile sperm from 10 to 58%, with non-progressive movement increasing from 9 to 19.3%. The number of spermatozoa with abnormal morphology doubled, and the DNA fragmentation index increased from 16 to 26%. Use of the swim-up procedure allowed us to sort progressively motile spermatozoa. The content of progressively motile spermatozoa in the samples obtained from the supernatant was 86%, which was 2.3 times higher than in frozen–thawed sperm (P≤0,01). The obtained results show the effective use of the swim-up procedure to determine the quality of semen in hybrid rams. These studies were carried out with financial support from the Russian Science Foundation, grant No. 18-16-00079 and the Ministry of Science and Higher Education of the Russian Federation.

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 48-49
Author(s):  
Elena Nikitkina ◽  
Artem Musidray ◽  
Anna Krutikova ◽  
Svetlana Timofeeva ◽  
Kirill Plemyashov ◽  
...  

Abstract Reindeer herding is a traditional activity of the indigenous peoples of the Arctic. Assisted reproductive technology in reindeer herding are under development. This is due to the difficulty of collecting reindeer sperm (Rangifer tarandus) in the Arctic. The aim of the work was to study the influence of age and rut period on the quality of reindeer (Rangifer tarandus) sperm. Semen was collected in Taimyr in autumn 2017, 2018. Ejaculate volume, total (TM) and progressive motility (PM) and sperm concentration were evaluated. TM, PM and sperm concentration were measured by CASA (computer-assisted semen analysis). Sperm was not found in caudae epididymidae in males at the age of six months. The sperm was collected only from caudae epididymidae from males aged 1.5 and 2.5 years. Collecting of ejaculated semen is possible in males from 3.5 years of age. The volume of ejaculate increased from 0.3±0.09 ml (mean±SD) at the age of 3.5 years to 0.7±0.09 ml at the age of 5.5 years and older (P < 0.05). Concentration of cells increased by 2 times in older males. It is known that reindeer sperm can be collected only during the rut. At the beginning and end of the rut, the collection of sperm by electroejaculation was not always successful, sperm was collected in 3 of 10 cases, while in the middle of the rut in 9 of 10 cases. There was no significant effect of the rut period on the ejaculate volume and sperm concentration. The lowest TM (51.1±2.16%) and PM (33.5±7.90%) were at the beginning of the rut, the highest at the end (73.0±5.02% and 59.0 ± 6,30%) (P < 0.01). Thus, the effect of age and rutting period on the reindeer sperm was observed. Authors acknowledge financial support from Russian Science Foundation, Grant No:17-16-01023.


2020 ◽  
Author(s):  
Yanling Zhao ◽  
Yaomei Wang ◽  
Feipeng Guo ◽  
Bo Lu ◽  
Jiale Sun ◽  
...  

Abstract Background: Tibetan pigs (TP) exhibit heritable adaptations to their hypoxic environments as a result of natural selection. Whereas, what candidate proteins affecting the sperm quality of boar on plateaus has not been clearly investigated yet. Methods: In this study, to reveal the candidate proteins affecting the quality of spermatozoa from boar on plateaus, we analyzed the sperm quality by Computer-assisted semen analysis (CASA) system and Reactive oxygen species (ROS) levels, compared the sperm proteomes between TP and Yorkshire pigs (YP) raised at high altitudes using the isobaric tags for relative and absolute quantitation (iTRAQ) in combination with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic method, and confirmed the relative expression levels of the four proteins by western blot. Results: The sperm quality of the TP was superior to that of the YP on plateaus. Of 1,555 quantified proteins, 318 differentially expressed proteins (DEPs) were identified. Gene ontology (GO) analysis revealed that the DEPs were predominantly associated with the sorbitol metabolic process, removal of superoxide radicals, cellular response to superoxide, response to superoxide and regulation of the mitotic spindle assembly. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were mainly enriched in pathways involved in the regulation of the actin cytoskeleton, glutathione metabolism, oxidative phosphorylation, and estrogen signaling. And based on the protein-protein interaction (PPI) network analysis, we identified 8 candidate proteins (FN1, EGF, HSP90B1, CFL1, GPX4, NDUFA6, VDAC2, and CP) that might play important roles that affect the sperm quality of boar on plateaus. Moreover, the relative expression levels of the proteins (CFL1, EGF, FN1, and GPX4) were confirmed by western blot. Conclusions: Our results reveal 8 candidate proteins (FN1, EGF, HSP90B1, CFL1, GPX4, NDUFA6, VDAC2, and CP) affecting the sperm quality of boar on plateaus, providing a reference for studies on improving sperm quality and the molecular breeding of boar on plateaus.


2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Y Cabell. Vives ◽  
P Belchin ◽  
C Lopez-Fernandez ◽  
M Fernandez-Rubio ◽  
J Guerrero-Sanchez ◽  
...  

Abstract Study question Is it useful to avoid sperm centrifugation in laboratory routine work to improve sperm quality and reproductive outcome in Assisted Reproduction Techniques (ART)? Summary answer Exclusion of sperm centrifugation for sperm selection using neat sperm samples (IO-lix), increases sperm quality in the collected subpopulation decreasing miscarriage rate after using ICSI. What is known already Inclusion of sperm centrifugation in ART is an aggressive intervention for sperm selection with ineludible production iatrogenic damage affecting sperm integrity. The application of IMSI, PICSI or microfluidic devices avoid sperm centrifugation and may improve the quality of the subsample obtained. However, these methodologies may result time consuming, expensive or producing poor results when the quality of the sperm is limited. We have already shown that a modified swim-up avoiding centrifugation (called IO-lix) is a low-cost and efficient alternative to microfluidic devices, recovers 100 times more concentration and reduces sperm DNA fragmentation with no significant differences to other methodologies. Study design, size, duration This is a retrospective study from 2018 to 2020 which includes patients with an average of age of 38.2 years using their own oocytes with ICSI as fertilization technique. Two aleatory groups of patients were made: Group 1: 88 cycles with 503 fertilized oocytes and 206 blastocysts were obtained with sperm samples processed by IO-lix and Group 2: 303 cycles, 1451 fertilized oocytes and 591 blastocysts using a standard “swim up” technique to process sperm. Participants/materials, setting, methods A total of 391 ICSI cycles were included in this retrospective study. The male factor was similar in both groups and they showed altered SDF previously to the cycle. We compared data of the motility and SDF of sperm samples before and after applying IO-lix and we analyzed by X2 contingence test differences on miscarriage rates between groups 1 and 2. Main results and the role of chance General sperm parameter changes after IO-lix showed that averaged sperm concentration observed in neat ejaculated samples was 62M/SD=46.4. Values obtained after IO-lix in the same samples were 12.3M/SD8.0. Averaged sperm motility in neat samples was 54%/SD=9.3 and 70.9%/SD=13.2 after IO-lix. Finally, sperm DNA fragmentation in neat samples was 35.8%/SD17.3, while these values decreased to 9.2%/SD=3.9 after IO-lix. About reproductive outcome results, significant differences were not obtained on the development to blastocyst stage rate comparing both groups (X2=0.003; p value = 0.954; Alpha 0.05). In the case of IO-lix processed samples, the pregnancy rate was 59.42% in Group 1 and 44.72% in Group 2 (X2=0.651; p value =0.419; Alpha 0.05). A total of 9 miscarriages of 41 clinical pregnancies (21.95%) were observed after IO-lix, while this number increases to 59 out of 123 clinical pregnancies, which means the 47.96% of the embryo transfers, when “swim-up” was used. In this case significant differences were obtained (X2=3.935; p value = 0.0.047; Alpha 0.05). Limitations, reasons for caution Being a pilot study aimed to understand the results of IO-lix in ART, correlations have not been stablished between the levels of sperm improvement after IO-lix and paired results of ART. This study would be necessary, specially to identify the possible origin of miscarriage associated to the male factor. Wider implications of the findings: Elimination of sperm centrifugation using a combined strategy of gradients and “swim-up” for sperm isolation, reduce miscarriage rate and produce equivalent results of blastocyst development to those obtained with “swim-up”. Being a cost-effective and improving laboratory workload, its use for sperm selection is recommended. Trial registration number Not applicable


2020 ◽  
Vol 32 (2) ◽  
pp. 137
Author(s):  
Y. Pirosanto ◽  
M. Valera ◽  
A. Molina ◽  
J. Dorado ◽  
S. Demyda-Peyrás

Inbreeding depression, a genetic condition produced by the mating of close-related individuals, has been associated with a reduction of fertility in several species. However, a loss in sperm quality was also associated with age. In horses, the few existing reports have described a tendency of both parameters to produce a negative effect on sperm quality. However, those reports were performed using a subjective evaluation of sperm motility. In the present study, a total of 692 ejaculates from 86 Pure Spanish stallions (PRE), aged between 3 and 22 years, were evaluated using a computer-assisted methodology to determine the effect of inbreeding in four semen parameters: free-gel volume (V), sperm concentration (C, by haemocytometer), and total (TM) and progressive (PM) sperm motility (by Spermvision sperm class analyser; Minitube). The inbreeding coefficient (F) was estimated using 300 000 PRE pedigree records approximately (minimum pedigree depth, eight equivalent complete generations; range, between 1 and 30.1%). Stallion, age, ejaculate, and season of semen collection were the variables included in the statistical model (general linear model), with ejaculate and season being the variables with a major effect (by variance components analysis). Our results showed that sperm concentration (r=−0.18; P<0.0001) and volume (to a lesser extent) were reduced with advancing age, both showing a major decline after 15 years of age. To the contrary, sperm motility was not affected by age of the stallion. We also found a negative correlation between the inbreeding coefficient and ejaculate volume (r=−0.14; P<0.001), with a marked decrease seen when F was between 7 and 20%. Also, a negative correlation was observed in PM (r=−0.08; P<0.05), although to a lower extent. Conversely, C and TM were not affected by inbreeding depression (P>0.05). In conclusion, our results demonstrated that high levels of inbreeding can compromise severely the sperm quality of the PRE stallion, which, subsequently, may have a negative influence on fertility. Ongoing studies using genomic data will help to detect genetic variants associated with stallion semen quality and how it is influenced by inbreeding in specific genomic regions.


2016 ◽  
Vol 28 (2) ◽  
pp. 221
Author(s):  
D. Le Bourhis ◽  
S. Camugli ◽  
P. Salvetti ◽  
L. Schibler ◽  
E. Schmitt

SensiTemp, a new in vitro maturation (IMV) bull straw concept, presents the advantage of colour changing while the straw is thawed. The colour of frozen straws is blue and straws start to become white when the temperature reaches 33°C, with a complete change of colour at 37°C. The objective of this study is to assess sperm quality after thawing of semen frozen in SensiTemp from 2 bulls, by analysing, in experiment 1, sperm motility and membrane integrity using computer-assisted semen analysis (CASA) and flow cytometry (FC), and, in experiment 2, the in vitro embryo production (IVP) using IVP technologies [IVM, IVF, and in vitro culture (IVC)]. The ejaculates of 2 bulls, selected during preliminary experiments on high in vitro fertility, were harvested at CIA L’Aigle, France, and split ejaculates were frozen in experimental (SensiTemp) and conventional (control) straws. In experiment 1 after thawing semen from the 2 types of straws (5 pooled straws each; 2 replicates), motility was assessed using the IVOS CASA system (Hamilton Thorne Inc., Beverly, MA, USA) and membrane integrity was evaluated through FC with Cytosoft software (Millipore-Guava Technologies Inc., Hayward, CA, USA). In experiment 2, IVF was used to evaluate the non-toxicity of SensiTemp and control straws. Cumulus-oocyte complexes (COC; n = 1178; 4 replicates) collected from slaughterhouse ovaries were matured in IVM medium (TCM-199 with bicarbonate, Sigma-Aldrich, Saint Quentin Fallavier, France; 10 µg mL–1 FSH-LH, Reprobiol, Liège, Belgium; and 10% FCS, Thermo Fisher, Illkirch, France) for 22 h. After fertilization, presumptive zygotes of each group (SensiTemp and control for each bull) were cultured in synthetic oviduct fluid medium (SOF, Minitube, Tiefenbach, Germany) with 1% estrous cow serum (ECS) and 0.6% BSA (Sigma-Aldrich, France) up to 8 days. All cultures were conducted at 38.5C in 5% CO2, and 5% O2. The cleavage and blastocysts rates were evaluated on Days 3 and 7, respectively, for each group. Embryo quality was recorded on Day 7 according to the IETS evaluation. Data from each bull were analysed separately using the chi-squared test (P < 0.05). In experiment 1, neither sperm motility from bull 1 (61.2 and 60.5%) and bull 2 (66.2 and 66.5%) nor membrane integrity from bull 1 (58.6 and 52.2%) and bull 2 (61.0 and 61.9%) were different between SensiTemp and control, respectively. Results from experiment 2 showed no difference (P > 0.05) in cleavage rate between SensiTemp and control for the 2 bulls: 92.1 and 91.7% for bull 1 and 94.2 and 94.6% for bull 2 respectively. The blastocysts rate on Day 7 did not differ (P > 0.05) among groups (47.5, 47.1 and 51.3, 50.4% for SensiTemp and control bull 1 and bull 2, respectively) nor the quality of embryos retrieved in the different groups: 25.4, 23.3, and 30.8, 29.6% in grade 1 embryo for SensiTemp and control bull 1 and bull 2, respectively. Those results demonstrate, in vitro, that the new SensiTemp straws were non-toxic and did not affect the semen quality after thawing nor did the SensiTemp straws affect the ability of sperm cells to fertilize oocytes and produce 8-day-old embryos.


2021 ◽  
Vol 27 (4) ◽  
Author(s):  
Julieta B Grosso ◽  
Luciana Zoff ◽  
Karina L Calvo ◽  
María Belén Maraval ◽  
Mariana Perez ◽  
...  

Abstract Decreased fertility is becoming an important social and medical problem and the male factor is involved in at least half of infertility cases. Since conventional semen analysis provides limited prediction of male fertility; in this work, we evaluated the potential use of seminal small RNAs (sRNA) as markers of semen quality in ART. Our bioinformatic analyses of available sRNA-seq databases showed that the most abundant sRNA species in seminal plasma of normozoospermic men are tRNA-derived fragments (tRFs), a novel class of regulatory sRNAs. These molecules not only exert their function within cells but also are released into the extracellular environment where they could carry out signaling functions. To evaluate whether the assessment of seminal tRFs in normozoospermic men has a predictive value for the clinical outcome in ART, we performed a prospective study with couples who underwent ICSI cycles with donated oocytes. The results obtained demonstrated that levels of 5’tRF-Glu-CTC, 5’tRF-Lys-CTT, and 5’tRF-Gly-GCC are significantly elevated in seminal samples from cases with repeated failed ICSI cycles, suggesting a potential association between increased seminal tRFs and unexplained male infertility. Interestingly, these tRFs showed a negative association with seminal testosterone, highlighting their involvement in male endocrinology. Our findings also suggest that tRFs could play a role in modulating male reproductive function in response to physiological stress since they showed significant associations with the levels of sperm DNA fragmentation in couples that achieved pregnancy but not in cases with failed ICSI cycles where seminal cortisol levels correlate with sperm quality.


Animals ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1973
Author(s):  
Muhammad Umair ◽  
Heiko Henning ◽  
Tom A. E. Stout ◽  
Anthony Claes

Sperm DNA fragmentation compromises fertilization and early embryo development. Since spermatozoa lack the machinery to repair DNA damage, to improve the likelihood of establishing a healthy pregnancy, it is preferable to process ejaculates of stallions with a high sperm DNA fragmentation index (DFI) before artificial insemination or intracytoplasmic sperm injection. The aim of this study was to examine a modified flotation density gradient centrifugation (DGC) technique in which semen was diluted with a colloid solution (Opti-prepTM) to increase its density prior to layering between colloid layers of lower and higher density. The optimal Opti-prepTM solution (20–60%) for use as the bottom/cushion layer was first determined, followed by a comparison between a modified sedimentation DGC and the modified flotation DGC technique, using different Opti-prepTM solutions (20%, 25% and 30%) as the top layer. Finally, the most efficient DGC technique was selected to process ejaculates from Friesian stallions (n = 3) with high sperm DFI (>20%). The optimal Opti-prepTM solution for the cushion layer was 40%. The modified sedimentation technique resulted in two different sperm populations, whereas the modified flotation technique yielded three populations. Among the variants tested, the modified flotation DGC using 20% Opti-prepTM as the top layer yielded the best results; the average sperm recovery was 57%; the DFI decreased significantly (from 12% to 4%) and the other sperm quality parameters, including progressive and total motility, percentages of spermatozoa with normal morphology and viable spermatozoa with an intact acrosome, all increased (p < 0.05). In Friesian stallions with high sperm DFI, the modified flotation DGC markedly decreased the DFI (from 31% to 5%) and significantly improved the other semen quality parameters, although sperm recovery was low (approximately 20%). In conclusion, stallion sperm DFI and other sperm quality parameters can be markedly improved using a modified flotation DGC technique employing a 40% Opti-prepTM cushion and a 20% top layer.


2021 ◽  
pp. 2473-2481
Author(s):  
Berlin Pandapotan Pardede ◽  
Tulus Maulana ◽  
Ekayanti Mulyawati Kaiin ◽  
Muhammad Agil ◽  
Ni Wayan Kurniani Karja ◽  
...  

Background and Aim: Protamine (PRM) is the major protein in the sperm nucleus and plays an essential role in its normal function. Moreover, PRM has great potential as a protein marker of semen production and quality. This study aimed to assess the potential of sperm bovine PRM as a protein marker of semen production and quality in bulls at the National Artificial Insemination (AI) Center of Indonesia. Materials and Methods: The semen production capacity of each bull was collected from frozen semen production data at the Singosari AI Center for 6 months, and was then divided into two groups (high and low). A total of 440 frozen semen straws from six Limousin (LIM), six Friesian Holstein (FH), six Peranakan Ongole (PO), and four Aceh bulls aged 4-5 years were used in the study. The frozen semen was used to measure the concentration of PRM1, PRM2, and PRM3 using the enzyme immunoassay method. The frozen semen was also used to assess the quality of the semen, including progressive motility (PM) through computer-assisted semen analysis, sperm viability through eosin–nigrosin analysis, and the DNA fragmentation index through Acridine Orange staining. Results: PRM1 was significantly higher in all bull breeds included in the study (p<0.00), followed by PRM2 (p<0.00) and PRM3 (p<0.00). PRM1 significantly affected semen production in LIM, FH, PO, and Aceh bulls (p<0.05). Moreover, PRM2 significantly affected semen production only in FH and Aceh bulls (p<0.05), whereas PRM3 affected this parameter in PO and Aceh bulls exclusively (p<0.05). Consistently and significantly, PRM1 was positively correlated with the PM and viability of sperm and negatively associated with its DNA fragmentation in LIM, FH, PO, and Aceh bulls (p<0.05; p<0.01). The correlation analysis between PRM2 and PRM3 and semen quality parameters varied across all bull breeds; some were positively and negatively correlated (p<0.05; p<0.01), and some were not correlated at all. Conclusion: PRM1 has excellent potential as a protein marker of semen production and quality in bulls at the National AI Center of Indonesia.


2012 ◽  
Vol 82 (6) ◽  
pp. 391-398 ◽  
Author(s):  
Barbara Wirleitner ◽  
Pierre Vanderzwalmen ◽  
Astrid Stecher ◽  
Dietmar Spitzer ◽  
Maximilian Schuff ◽  
...  

Background: This study aimed to investigate the influence of an oral antioxidative supplementation on sperm quality of in vitro fertilization (IVF) patients, as analyzed by sperm motility according to the WHO criteria and motile sperm organelle morphology examination (MSOME). Methods: Semen samples were collected from 147 patients before undergoing an IVF/intracytoplasmic morphologically-selected sperm injection (IMSI) cycle and 2 - 12 months after an antioxidative supplementation. Semen analysis was evaluated according to WHO and MSOME criteria. Spermatozoa were grouped according to the size of nuclear vacuoles within the sperm’s heads. Patients were divided into oligoasthenoteratozoospermic (OAT) and non-OAT men. Between first and second semen analysis, patients were supplemented orally with an antioxidative preparation. Results: After the antioxidative therapy we observed a significant reduction in the percentage of immotile sperm cells in the patients. Additionally, the percentage of class I spermatozoa according to MSOME criteria was significantly higher after antioxidative supplementation. In OAT patients the percentage of class I sperm was found to be increased, although not significantly. However, we observed a drastic improvement in sperm motility as well as in total sperm count in this group. Conclusion: The results demonstrated a considerable improvement in semen quality, notably in OAT patients. Considering the putative relationship between semen quality on the one hand and reactive oxygen species on the other, the observed changes in the sperm parameters indicate that a decline in semen quality, and even subtle morphological changes, might be associated with oxidative stress. Our findings suggest that an antioxidative and micronutrient supplementation has a remarkable benefit for IVF patients having restricted sperm parameters, in particular.


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