Investigating mutations in the genes GDF9 and BMP15 in Pelibuey sheep through the amplification-refractory mutation system with tetra-primers

Single Nucleotide Polymorphisms (SNP) or mutations are variations with a broad distribution in the genome and, as part of genetic studies, SNP allow the identification of allelic variants related to characteristics of economic importance in sheep production. However, the identification of SNP and their genotypes through sequencing is expensive, as it requires specialized materials and equipment. The objective of this study was to identify polymorphisms and their genotypes in the growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes in Pelibuey sheep using the tetra-primer amplification-refractory mutation system through polymerase chain reaction (T-ARMS-PCR). DNA extraction and amplification of BMP15 and GDF9 were conducted from blood samples contained in WhatmanTM FTATM cards from 60 multiparous Pelibuey ewes with reproductive records. The T-ARMS-PCR methodology allowed the identification of wild-type genotypes and mutated homozygous genotypes in polymorphisms G4 and G6 of GDF9, whereas mutations in the BMP15 gene were not found. These results were confirmed by sequencing. In conclusion, the T-ARMS-PCR methodology allowed the identification of mutated and wild-type genotypes in SNP G4 and G6 of GDF9, although no mutations were found in BMP15 in Pelibuey sheep. This technique was found to be reliable, rapid, and easily applied to identify polymorphic genotypes

Use of Andromed® and OviXcell® diluents in the processing of sheep semen with the addition of HTF

Objective: To assess the effect of two ovine semen diluents, Andromed® and OviXcell®, on the mass motility, vitality, and acrosomal integrity of spermatozoa processed at different temperatures and with the addition of post-thawed HTF (human tubal fluid). Design/Methodology/Approach: Semen from 2 Dorper ram was used. Four ejaculations per male were collected and diluted with Andromed® and Ovixcell®. There were four treatments with Ovixcell®: 1) fresh Ovixcell®, 2) refrigerated Ovixcell®, 3) post-thawed Ovixcell®, and 4) post-thawed Ovixcell® + HTF. Four treaments with Andromed® were also carried out: 1) fresh Andromed®, 2) refrigerated Andromed®, 3) post-thawed Andromed®, and 4) post-thawed Andromed® + HTF. Mass motility, vitality, and acrosomal integrity were performed with a CASA computer system and statistically analyzed with the GLM procedure of the SAS software. Results: When the effect of Ovixcell® mass motility was assessed, the following results were obtained, with differences (p˂0.05) between treatments: 87% in fresh semen, 72% refrigerated semen, 55% in post-thawed semen, and 68% in post-thawed semen + HTF. A clear difference (p˂0.05) was observed when HTF was added to post-thawed semen (13%). Andromed® behaved in the same way as Ovixcell® (p˂0.05) and a 18% recovery was observed with the addition of HTF. A high percentage of live spermatozoa with intact acrosome was observed for fresh semen (97.8%), while it diminished (p<0.05) as the temperature of refrigerated and frozen semen gradually decreased. Study Limitations/Implications: More experimental units should be used, despite the increase in maintenance costs per animal. Findings/Conclusions: The use of Andromed® and OviXcell® diluents was satisfactory in relation to the mass motility, vitality, and acrosomal integrity in spermatozoa processed at different temperatures (fresh, refrigerated, and post-thawed). Furthermore, the addition of HTF increased post-thawed mass motility

Utjecaj trajanja in vitro sazrijevanja i inkubacije s aktivirajućim čimbenikom na kapacitet izlijeganja goveđih partenota - kratko priopćenje

The period of both in vitro maturation (IVM) and incubation with oocyte activators affects the blastocyst yield following parthenogenetic activation (PA). Nevertheless, it is still unknown how these conditions impact the expansion and hatching rates of bovine parthenogenetic blastocysts. The objective of this study was to assess the influence of the duration of IVM and exposure to the activating agent, 6-dimethylaminopurine (6-DMAP), on a number of developmental parameters in bovine parthenotes, including: Cleavage, blastocyst formation, expansion, and hatching. Slaughterhouse oocytes were subjected to different periods of IVM. Subsequently, eggs were first parthenogenetically activated for five minutes with ionomycin and then incubated for distinct lengths of time with a second activator, 6-DMAP. The treatments were: a) Control: 22 h IVM/4 h 6-DMAP; b) 22 h IVM/5 h 6-DMAP; c) 24 h IVM/4 h 6-DMAP; and d) 24 h IVM/5 h 6-DMAP. Developmental stages were evaluated at day 4 and day 8 of in vitro culture (IVC). No differences were detected in most developmental parameters. However, the duration of IVM and incubation with 6-DMAP significantly affected (P<0.05) hatching capacity considering the number of blastocysts (Hatch./Blast.). Also, this same variable was higher (P<0.05) in group b) 22 h IVM/5 h 6-DMAP (45.89 ± 12.59%), as compared to c) 24 h IVM/4 h 6-DMAP (6.67 ± 6.67%). In conclusion, the length of IVM and incubation with 6-DMAP influenced parthenogenetic development, where 22 h IVM/5 h 6-DMAP was the condition producing the highest Hatch./Blast. rate in bovine parthenotes.Vrijeme in vitro sazrijevanja (IVM) i vrijeme inkubacije s aktivatorima oocista utječu na stvaranje blastocista nakon partenogenetske aktivacije (PA). Ipak, još uvijek se ne zna kako navedeno utječe na ekspanziju i stopu izlijeganja goveđih partenogenetskih blastocista. Cilj rada bio je istražiti utjecaj trajanja IVM i izloženosti aktivirajućem čimbeniku 6-dimethylaminopurinu (6-DMAP) na više razvojnih parametara u goveđih partenota, uključujući diobu, formiranje blastociste, ekspanziju i izlijeganje. Oocite prikupljene u klaonicama bile su podvrgnute različitom trajanju IVM. Nakon toga jajašca su prvo partenogenetski aktivirana s ionomicinom kroz 5 minuta i nakon toga inkubirana tijekom određenih vremenskih razdoblja sa drugim aktivatorom, 6-DMAP. Protokoli po istraženim skupinama bili su sljedeći: a) kontrolna skupina 22 h IVM/4 h 6-DMAP, b) skupina 22 h IVM/5 h 6-DMAP, c) skupina 24 h IVM/4 h 6-DMAP i d) skupina 24 h IVM/5 h 6-DMAP. Razvojni stadiji in vitro kulture (IVC) procijenjivani su 4. i 8. dan. Za većinu razvojnih parametara nisu utvrđene razlike između istraženih skupina. Ipak, trajanja IVM i inkubacije sa 6-DMAP znakovito su utjecali (P<0,05) na kapacitet izlijeganja kad se u obzir uzme broj blastocista (izlijeganja/ blasociste). Također, isti pokazatelji bili su viši (P<0,05) u skupini b) 22 h IVM/5 h 6-DMAP (45,89 ± 12,59 %) u odnosu na skupinu c) 24 h IVM/4 h 6-DMAP (6,67 ± 6,67 %). Zaključno, trajanje IVM i inkubacije sa 6-DMAP utjecali su na partenogenetski razvoj, pri čemu je 22 h IVM/5 h 6-DMAP kombinacija koja u goveđih partenota proizvodi najvišu stopu za pokazetelj izlijeganje/blasociste

Increase of the motility of buck semen with Andromed® extender and the addition of HTF

Objective: To test whether HTF (Human Tubal Fluid) is capable of protecting goat sperm cells increasing the semen m Objective: To determine if HTF (Human Tubal Fluid) can protect buck sperm cells, increasing semen motility when used by itself or combined with the Andromed® commercial extender. Design/Methodology/Approach: The semen of eight bucks of the Boer breed was used (10 ejaculates per male). Each ejaculate was diluted in three treatments (5 repetitions per treatment): 1) only Andromed®; 2) only HTF; and 3) Andromed® + HTF. The following variables were evaluated: mass motility (MM) and progressive motility (MP) in fresh (37 °C) and refrigerated (5 °C) semen. The differences between treatments were detected, based on the MM and MP in fresh and refrigerated semen variables, using the GLM procedure (SAS V9). In the case of the MM evaluated in three different periods, the measures repeated over time were analyzed using the Proc Mixed procedure (SAS V9). Results: Regarding the quality of fresh spermatozoa, MM and MP recorded similar values (p&gt;0.05) for the three treatments. On the one hand, MM registered 97.1% for Andromed®, 95.8% for HTF, and 97.5% for Andromed® + HTF; its highest value was achieved with the Andromed® + HTF combination, followed by Andromed® and HTF. On the other hand, the highest MP percentage was observed with the combination of Andromed® + HTF (91.0%), followed by Andromed® (90.4%), and HTF (89.0%). Regarding the quality of the sperm refrigerated at 5 °C, the combination of Andromed® + HTF had a higher MP value (p&lt;0.05) than other treatments. Study Limitations/Implications: In all the evaluated treatments, the motility of sperm decreased over time (0 to 24 hours after cooling); however, the treatments with Andromed®, and Andromed®+HTF maintained a high motility. Findings/Conclusions: The combination of Andromed® and HTF can be used to dilute both fresh and cooled semen

Reproductive and productive behavior of hair sheep under an intensive production system

Objective: To evaluate the reproductive and productive behavior of four hair sheep breeds in an intensive production system. Design/Methodology/Approach: Data recorded over 4 years (2016-2019) from a production unit that works with Blackbelly, Pelibuey, Dorper, and Katahdin breeds were analyzed. The following variables were evaluated: fertility, prolificacy, birth weight (BW), weaning weight (WW), weaned lamb yield (WLY), daily weight gain during lactation (DWGL), offspring born alive (OBA), male offspring (MO), and female offspring (FO). All information was analyzed using Chi-square tests and analysis of variance. Results: The fertility, BW, WW, and WYL variables recorded different values between breeds (P&lt;0.05). The greatest fertility and prolificacy were recorded by Blackbelly, while Dorper recorded the lowest values (P&lt;0.05). BW and WW were highest in Dorper and lowest in Blackbelly (P&lt;0.05). Finally, the sheep that recorded the highest WYL were the Blackbelly, while the Dorper registered the lowest values (P&lt;0.05). BW and DWGL values reached maximum values (P&lt;0.05) in Dorper rams, followed by Katahdin. The greatest number of OBA was observed in Blackbelly and Pelibuey (P&lt;0.05). Study Limitations/Implications: Further studies should be carried out to validate this research. Findings/Conclusions: Blackbelly ewes produced more kilograms of weaned lambs per lambing ewe. The Dorper and Katahdin rams in the pre-weaning period recorded higher weight at weaning

Effect of the breed, reproductive season, doses and time of application of eCG in the estrous cycle in hair ewe lambs

Objective: to evaluate the effect of breed, reproductive season, dose and time of application of equine chorionic gonadotropin (eCG) on the estrous cycle and ovarian activity in hair ewe lambs. Design/methodology/approach: 216 Hair lambs were used (62 Dorper, 69 Katahdin y 85 Pelibuey), 91 in the high reproductive season and 125 in the low reproductive season, they were synchronized with intravaginal sponges containing 20 mg of fluorogestone acetate (FGA) and equine chorionic gonadotropin (eCG; 200 and 300 IU) intramuscularly. The treatments are the breed, reproductive season, dose and time of application of eCG. The presence of estrus was analyzed under a logistic regression model, the interval to estrus and ovulation rate were analyzed by analysis of variance using a completely randomized design with a 2x2x2x3 factorial arrangement with the PROC LOGISTIC and PROC GLM procedures. Results: the breed influenced (P&lt;0.01) the presence of estrus, Dorper ewe lambs presented 9.74 times more possibilities than the Pelibuey. The interval to estrus was shorter in duration (P&lt;0.05) in Dorper (29.5±0.9 h) and Katahdin (29.1±0.9 h) compared to Pelibuey (34.8±0.9 h). The interval to estrus was lower (P&lt;0.05) with the application of 200 or 300 IU of eCG 24 h before the end of the protocol, compared to the application of 200 IU of eCG at the time of progestogen withdrawal. Ovulation rate was only affected by breed (P&lt;0.05), being higher in Pelibuey (2.4±0.1) compared to Dorper (2.0±0.1) and Katahdin (1.9±0.1). Study limitations/implications: To complement the study, it would be convenient to take the research to a second phase where the gestation part of the females is included to relate it to the ovulation rate, and also to make measurements of ovarian structures by means ultrasound. Findings/conclusions: Estrus and ovarian activity in hair ewe lambs synchronized with progestogens is mainly influenced by breed

Cultivo de embriones a través de un medio modificado para aumentar la preñez en ganado bovino

Las condiciones de cultivo in vitro mejoran la calidad del embrión durante el desarrollo temprano y tardío, y modificaciones en las condiciones de cultivo pueden aumentar la expresión génica, el metabolismo celular o el estado de la impresión genómica. La investigación se enfocó en el metabolismo del embrión que fue medido en un medio modificado describiendo las principales vías por las cuales se implementa el Adenosin Trifosfato. Los medios usados para la capacitación espermática fueron HTF QUEEN, HTF HEPES, SPERM WASH MEDIUM, también se utilizó ATP. El espermatozoide fue incubado para la fertilización in vitro junto con los medios modificados para luego transferir los embriones a las vacas receptoras. El embrión tuvo un mejor desarrollo en presencia del ATP que con los medios estándares usados, preñándose 2 de las 5 vacas