scholarly journals Genetic diversity in oocyte donors used in in vitro bovine embryo production programs in Brazil

2019 ◽  
Vol 32 (2) ◽  
pp. 90-99
Author(s):  
Wilder Hernando Ortiz Vega ◽  
Celia Raquel Quirino ◽  
Aylton Bartholazzi Junior ◽  
Clara Slade Oliveira ◽  
Raquel Varella Serapião ◽  
...  

Background: Current reproductive management of bovine elite populations involves the use of assisted reproductive technologies (ARTs), aiming to obtain the greatest genetic gain. However, inadequate use of ARTs may lead to loss of genetic diversity in the offspring. Objective: To assess the genetic diversity in elite female cattle populations used in commercial in vitro embryo production. Methods: Using genetic and ecological approaches for the study of populations based on microsatellite markers, we assessed the genetic diversity between and within populations of cows used in commercial in vitro embryo production programs in Brazil. Results: Endogamy within populations varied from zero to 9.1%, while heterozygosity between populations (FST) was <0.05 in the different population interactions. AMOVA showed 1% variation between populations, 8% between individuals and 91% within individuals. The dimensionality reduction method utilized indicated a lack of structure in the populations analyzed, identifying two main clusters in the three populations. Conclusions: Low genetic diversity between cow populations associated with commercial programs of in vitro embryo production in Brazil was evidenced. Variable levels of endogamy within the populations were observed. Approaches of population genetics as well as ecological diversity can be implemented to more thoroughly estimate genetic diversity in livestock populations.Keywords: allele frequencies, heterozygosity, inbreeding, microsatellite markers, oocyte. ResumenAntecedentes: El actual manejo reproductivo en poblaciones de bovinos de élite incluye la utilización de tecnologías de reproducción asistida (ARTs) con el fin de obtener mayor ganancia genética. Sin embargo, el uso inadecuado de las ART puede llevar a la pérdida de diversidad genética en los descendientes. Objetivo: Evaluar la diversidad genética en poblaciones de vacas de élite utilizadas en la producción comercial de embriones bovinos in vitro. Métodos: Utilizando abordajes de la genética y ecología de poblaciones basados en marcadores microsatélites, evaluamos la diversidad genética entre y dentro de poblaciones de vacas participantes de programas comerciales de producción de embriones in vitro en Brasil. Resultados: La endogamia dentro de las poblaciones varió de cero a 9.1%, mientras que la heterocigosidad entre poblaciones (FST) fue <0.05 en las diferentes interacciones de la población. El AMOVA mostró variación del 1% entre poblaciones, 8% entre individuos y 91% dentro de individuos. El método de reducción de dimensionalidad utilizado indicó una falta de estructura en las poblaciones analizadas, identificando dos grupos principales en las tres poblaciones. Conclusiones: Se evidenció una baja diversidad genética entre las poblaciones de vacas asociadas a programas comerciales de producción de embriones in vitro en Brasil. Se evidenciaron niveles variables de endogamia entre las poblaciones. Abordajes de la genética poblacional, así como de diversidad ecológica pueden ser implementados para estimar de manera más amplia la diversidad genética en poblaciones animales de interés pecuario.Palabras clave: endogamia, frecuencia alélica, heterozigosidad, marcadores microsatélites, ovocito. ResumoAntecedentes: O atual manejo reprodutivo das populações de elite em bovinos envolve o uso de tecnologias de reprodução assistida (ARTs), visando obter o maior ganho genético. No entanto, o uso inadequado de ARTs pode levar à perda de diversidade genética na prole. Objetivo: Avaliar a diversidade genética em populações de vacas de elite utilizadas na produção comercial de embriões bovinos in vitro. Métodos: Utilizando abordagens da genética e ecologia de populações baseadas em marcadores microssatélites, foi avaliada a diversidade genética entre e dentro das populações de vacas participantes de programas comercias de produção in vitro de embriões. Resultados: A endogamia dentro das populações variou de zero a 9,1%, enquanto a heterozigosidade entre populações (FST) foi <0,05 nas diferentes interações populacionais. AMOVA mostrou variação de 1% entre populações, 8% entre indivíduos e 91% dentro de indivíduos. O método de redução de dimensionalidade utilizado indicou uma falta de estrutura nas populações analisadas, identificando dois clusters principais nas três populações. Conclusões: Baixa diversidade genética entre populações de vacas associadas a programas de produção in vitro de embriões foi evidenciada. Níveis de endogamia variáveis dentro das populações foram observados. Abordagens da genética populacional assim como de diversidade ecológica podem ser implementadas na tentativa de estimar de maneira mais abrangente a diversidade genética em populações animais de interesse pecuário.Palavras–chave: endogamia, frequência alélica, heterozigosidade, marcadores microssatélite, oócito.

Animals ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 3319
Author(s):  
Morgan F. Orsolini ◽  
Stuart A. Meyers ◽  
Pouya Dini

As the use of assisted reproductive technologies (ART) and in vitro embryo production (IVP) expand in the equine industry, it has become necessary to further our understanding of available semen selection techniques. This segment of our two-section review will focus on the selection of spermatozoa based on quality and sex for equine intracytoplasmic sperm injection (ICSI), as well as current and future developments in sperm sorting technologies. Ultimately, novel methods of semen selection will be assessed based on their efficacy in other species and their relevance and future application towards ARTs in the horse.


2013 ◽  
Vol 25 (1) ◽  
pp. 267
Author(s):  
N. Y. Rho ◽  
F. A. Ashkar ◽  
T. Revay ◽  
P. Madan ◽  
W. A. King

Thyroid hormones (TH) play an important role in the physiology of vertebrates, ranging from the regulation of metabolic processes to cell proliferation, differentiation, and embryo development. We have previously shown a beneficial effect of supplementing TH in in vitro embryo production media. Recently, detection of TH receptors (TR) in oocytes and early stages of pre-implantation embryos indicated a possible regulatory role for TH in these stages (unpublished data). The objective of this study was to investigate the importance of TR expression in the pre-attachment bovine embryo in vitro. Bovine embryos, produced by standard in vitro embryo production procedures, were microinjected at the zygote stage with small interfering RNA (siRNA) specifically designed for knocking down either TR-α or TR-β. In addition, groups of zygotes were microinjected with scrambled siRNA (SI) or were not injected (NI), and these groups served as controls. Embryo developmental rates were assessed using light microscopy for blastocyst formation rates and expression of TR messenger RNA (mRNA) transcripts at the blastocyst stage was assessed by quantitative PCR across all groups. Expression of TR mRNA was normalized against glyceraldehyde 3-phosphate dehydrogenase, H2a, and 18S as reference genes. There was a significant decrease in blastocyst formation rates in both embryo groups injected with either TR-α (P < 0.002) and TR-β (P < 0.001) siRNA compared with the NI and SI groups. Moreover, the TR-β knockdown group exhibited a lower developmental rate than the TR-α knockdown group, which indicates a stronger inhibitory role for TR-β. Quantification of the level of TR mRNA expression in four groups normalized with three different reference genes shows a consistent significant reduction in the levels of TR-α (P < 0.05) and TR-β (P < 0.02) mRNA transcripts compared with the NI and SI groups. However, TR-β expression was inhibited more than was TR-α expression. In conclusion, the results indicate that knocking down either TR-α or TR-β restrains embryo development. This suggests that TH play a vital role in the regulation of embryo development through their receptors during bovine early embryogenesis. The specific role of each of these receptors and their mechanism of action in mediating development needs to be further elucidated. Funding was provided by CRC, NSERC, and the EmbryoGENE network.


2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 37-37
Author(s):  
Nicola Oosthuizen ◽  
Pedro Levy Piza Fontes ◽  
G Cliff Lamb

Abstract Bos indicus and Bos taurus cattle diverged from an evolutionary standpoint more than 110,000 years ago. Since then, Bos indicus cattle have undergone genetic adaptations beyond the commonly discussed increased thermo-tolerance and parasite resistance. Several physiological differences exist between Bos indicus and Bos taurus cattle, and it is important to consider these differences when establishing reproductive management strategies. It has been well described that Bos indicus cattle have a delayed onset of puberty and longer periods of postpartum anestrus, yet through the utilization of estrus synchronization protocols these challenges can be attenuated. However, when Bos indicus females are exposed to these protocols, they are known to have smaller dominant follicles, lower expression of estrus, and decreased pregnancy rates to artificial insemination (AI) when compared to Bos taurus females. These factors can be overcome through the utilization of estradiol and progesterone based synchronization protocols, which improve follicular dynamics and yield acceptable pregnancy rates to assisted reproductive technologies in cattle adapted to tropical or subtropical conditions. However, the use of estrogens for synchronization purposes is not permitted in the United States, and cattle producers need to rely on GnRH-based protocols. Another key difference between subspecies, is that Bos indicus females have greater antral follicle counts than Bos taurus females, which proves beneficial for in vitro embryo production. Therefore, an opportunity exists to explore the greater productivity of donors in embryo production in order to improve genetics in herds that utilize these breeds.


2005 ◽  
Vol 17 (2) ◽  
pp. 273 ◽  
Author(s):  
Y. Huang ◽  
X. Zhang ◽  
B. Gasparrini ◽  
G.A. Presicce

In this preliminary study, in vitro embryo production and cryopreservation in two river type buffaloes (Murrah and Nili-Ravi) imported into China have been carried out. The objective of the study was enhancement of the genetic merit and productive performances of imported river buffaloes in conjunction with the utilization of local swamp buffaloes. In order to improve milk and meat production in China local swamp buffaloes (2n = 48), which are the predominant subspecies, have been crossbred with imported river buffaloes (Murrah and Nili-Ravi: 2n = 50). At present, several hundred thousand crossbred heads have been produced, and although both males and females can reproduce with 2n = 49 crossbred buffaloes, their reproductive performances are significantly reduced when compared to 2n = 50 buffaloes. As an alternative approach, a program of embryo production in river buffaloes and transfer into both river and swamp buffaloes has been implemented at the Guangxi Buffalo Research Institute, in Nanning, P.R. China. Some preliminary results are presented: from a start-up experiment, a total of 46 river buffaloes were subjected to 2 to 3 ovum pickup sessions at 4-day intervals. A total of 750 antral follicles were punctured and 495 (66%) cumulus-oocyte complexes (COCs) were retrieved. Only COCs characterized by at least one layer of granulosa cells (n = 451; 91.1%) were considered for in vitro maturation (IVM). COCs were matured in TCM 199 + 10% FCS, 0.5 μg/mL FSH, 5 μg/mL LH, and 1 μg/mL estradiol in the presence of cysteamine (50 μM) at 39°C under 5% CO2 in humidified air for 24 h. Of the initial 451 COCs matured, only 277 could be considered for in vitro fertilization (IVF). IVF was performed at 39°C under CO2 in humidified air in TALP medium supplemented with 0.2 mM penicillamine, 0.1 mM hypotaurine, and 0.01 mM heparin. Frozen/thawed sperm from a tested bull was treated by swim-up procedure and used at a final concentration of 20 million/mL. Following 20 to 22 h of co-incubation, presumptive zygotes were cultured in SOF medium, supplemented with essential and non-essential amino acids and 8 mg/mL BSA, in a gas atmosphere of 5% CO2, 7% O2, and 88% N2. A total of 41 (14.8%) blastocysts were produced, of which 33 were vitrified and 8 transferred immediately into available swamp and river buffalo recipients. Two calves were born (25%) from the transfer of fresh embryos into one river and one swamp buffalo. In vitro embryo production in the buffalo species is still characterized by a high degree of variable results. However, these preliminary results reinforce the need to implement newly developed reproductive technologies not only for speeding up genetic gain of already productive species, but also for the utilization of local breeds characterized by reduced productive performance.


2012 ◽  
Vol 24 (1) ◽  
pp. 210
Author(s):  
S. M. Bernal ◽  
J. Heinzmann ◽  
D. Herrmann ◽  
A. Lucas-Hahn ◽  
B. Timmermann ◽  
...  

Bovine oocytes and embryos have been established as a valuable model for studying human early development, specifically after assisted reproductive technologies (ART). Efforts for the improvement of ART in the last years have focused on culture media and conditions. Recently, Albuz et al. (2010) reported that the culture of bovine cumulus–oocyte complexes (COC) with cyclic adenosine 3′, 5′-monophosphate (cAMP) modulators, before and during extended in vitro maturation (IVM), improved blastocyst quality and yields in mice and cattle. In this study, we investigated the influence of an extended IVM phase in combination with cAMP modulators on blastocyst yields and quality, the effects on mRNA expression profiles and epigenetic marks. We compared these results to the standard protocol (Wrenzycki et al., 2001) used in our laboratory with oocytes from different retrieval methods. Oocytes were retrieved from slaughterhouse ovaries either by slicing or follicular aspiration. The COC were either subjected directly to IVM using the standard TCM-based protocol for 24 h (TCM24-slicing and TCM24-aspiration, respectively) or oocytes that were retrieved by aspiration were treated with forskolin and IBMX for a 2-h pre-IVM period, followed by an extended IVM phase of 30 h in TCM, supplemented with cilostamide (cAMP30-aspiration). Statistical analyses were performed using 1-way ANOVA followed by the nonparametrical Kruskal–Wallis test. Maturation rates were 79.3 ± 2.6% in TCM24-aspiration, 74.2 ± 8.8% in cAMP30-aspiration and 70.4 ± 5.1% in TCM24-slicing oocytes. Matured oocytes were fertilized in vitro with semen from a bull previously proven to be suitable for IVF. Blastocyst rates from presumptive zygotes were significantly higher (P = 0.003) in the TCM24-aspiration group (32 ± 7%) compared to TCM24-slicing (23 ± 7%) and cAMP30-aspiration (22 ± 5%). Analysis revealed that cell numbers were rather similar in the 3 experimental groups (125 ± 19, 128 ± 15 and 129 ± 9), while in vivo-produced blastocysts possessed slightly more cells (134 ± 17; P ≥ 0.05). RT-qPCR analysis of mRNA expression for a panel of genes indicative of embryo quality including DNMT3a, SLC2A8, COX2 and PCK2, showed that blastocysts derived from both aspiration protocols were similar to in vivo embryos, but were different from blastocysts resulting from the ovary-slicing protocol. Specifically, the expression profile of COX2, which is involved in pregnancy outcome and in the response to growth factors, indicates an enhanced developmental competence of aspirated oocytes. However, the transcript level of EGR1 (early growth response) was significantly higher (P = 0.009) in in vivo-derived blastocysts in comparison to all in vitro treatments. The investigation of the epigenetic status of the in vitro-derived blastocysts based on bisulfite sequencing of 2 satellite repeat sequences is currently underway. Results so far indicate that the method of obtaining the oocytes (slicing vs aspiration) for in vitro production of bovine embryos is of greater influence on blastocyst quality than IVM conditions.


2009 ◽  
Vol 21 (1) ◽  
pp. 161
Author(s):  
A. M. Paprocki ◽  
C. M. Syverson ◽  
R. W. Koppang ◽  
J. R. Dobrinsky

Although in vivo matured, ovulated, or both, oocytes provide the finest genetic material for use in assisted reproductive technologies (ART), their en masse production requires livestock production facilities, staff and associated overhead, is expensive and labor intensive, their harvest involves surgical or laparoscopic expertise, and large yields needed for en masse daily embryo production are cumbersome and very costly. In vitro-matured (IVM) oocytes have long been a practical gamete source for ART, including in vitro fertilization, ICSI and cloning. Rather than using conventional IVF to produce embryos, we employ in vitro oocyte activation for the production of diploidized parthenogenetic embryos, removing problems associated with variable embryo production due to polyspermic inseminations. In this way, we can produce a repeatable and consistent supply of mature oocytes, advanced embryos, or both, used in product testing, quality control, transgenic or cloned (or both) embryo production, in vitro development controls, as well as in-house culture control embryos for customer scientific data sharing. In this study, we observe mature oocyte and parthenogenetic embryo production over a complete year as control information for our laboratory. Additionally, colleagues may use these data for comparison in their own scientific mission. At least 3 times a month for 12 consecutive months, ovaries were collected from mature females at an abattoir and transported to our laboratory. Cumulus–oocyte complexes were aspirated from 4–6 mm follicles with an 18-gauge needle fixed to a vacuum pump system. Only COC surrounded by two or more layers of compact cumulus investment and containing oocytes of equal size were placed into a commercial TCM-199-based IVM system (Minitube of America Inc., Verona, WI, USA). After 42 h IVM, mature oocytes were isolated from their expanded cumulus and subjected to chemical (ionomycin/DMAP) parthenogenetic activation based on US Patent 5,496,720. Embryos were cultured 120 h in NCSU-23, then cultured for an additional 48 h in NCSU-23 (no BSA) supplemented with 10% FBS. A minimum of 1504 premium and 4604 standard oocytes (Minitube of America Inc.) were placed into IVM. Both premium (1364, 90.7%) and standard (4061, 88.2%; P > 0.05) oocytes are used to produce mature oocytes (MO). Of 781 premium MOs made into diploidized parthenogenetic embryos, 459 (58.8%) developed into blastocysts (61.3 cells/embryo). Of 2068 standard MO made into diploidized parthenogenetic embryos, 914 (44.2%; P < 0.05) developed into blastocysts (64.7 cells/embryo). En masse in vitro maturation of oocytes can supply a repeatable and consistent supply of mature oocytes for use in assisted reproductive technologies. These MO have the developmental potential to form blastocysts in vitro and enable scientists to infer developmental competence of in vitro-produced embryos for research and commercial use.


2019 ◽  
Vol 31 (1) ◽  
pp. 197
Author(s):  
L. K. Hatamoto-Zervoudakis ◽  
M. F. Duarte Jr ◽  
T. F. Motheo ◽  
P. P. Tsuneda ◽  
J. T. Zervoudakis

Cottonseed and its derivatives are frequently used in cattle feed as an effective dietary fibre supply and high protein and energy food source. However, the cotton plant contains gossypol, which in its free form induces male and female infertility. Therefore, this study aimed to evaluate the effect of gossypol supplementation on bovine in vitro embryo production. Ovaries were retrieved from slaughterhouses, and cumulus-oocyte complexes (COC) were recovered by follicular puncture. Based on free gossypol concentration present on the in vitro maturation, sperm capacitation, IVF and in vitro culture media, grades I, II and III COC (n=646) were divided in 3 treatments: 0μg mL−1 (control), 5μg mL−1 (G5) and 10μg mL−1 (G10). The COC were matured under a humidified atmosphere of 5% CO2 in air at 38.5°C for 24h in 90-μL droplets containing TCM-199 supplemented with 10% FCS, 0.2mM sodium pyruvate, LH, FSH, 75μg mL−1 amikacin, 17β-oestradiol. Each droplet corresponded to one replicate (n=14) and contained 15 to 18 COC. Matured COC and sperm were co-incubated in droplets (8-13 COC per 90μL) of TALP-IVF media supplemented with 6mg mL−1 BSA, 0.2mM sodium pyruvate, 30μg mL−1 heparin, 20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine, 75μg mL−1 amikacin under a 5% CO2 humidified atmosphere at 38.5°C, for 20h. For IVF, non-sexed frozen-thawed semen was selected with Percoll® gradient. The resulting pellet was subjectively evaluated for motility and concentration and then diluted to final concentration of sperm mL−1 with fertilization medium. Presumptive zygotes were then cultured in 90-μL droplets of SOFaaci medium supplemented with 2.7mM myo-inosytol, 0.2mM pyruvate, 2.5% FCS (v/v), 5mg mL−1 BSA, 75μg mL−1 amikacin, and maintained for 8 days at 38.5°C in a humidified atmosphere with 5% CO2 in air. Cleavage, blastocysts production and hatching rates were evaluated at Days 3, 7 and 8, respectively. Data were submitted to ANOVA for parametric data and Wilcoxon test for non-parametric variables using the SAS software (SAS Institute Inc., Cary, NC, USA). Significance level was set at 5%. Cleavage rates of the control (81.05%) and G5 (71.85%) were higher compared with G10 (19.64%; P &lt; 0.0001). Blastocyst production was lower in G5 (12.18%) compared with control (30.35%), and the addition of 10μg mL−1 of free gossypol (G10) completely inhibited embryo development (0%; P &lt; 0.0001). As for the percentage of hatched blastocysts, the control (66.75%) had greater values compared with G5 (34.52%; P &lt; 0.0001). Thus, the addition of 5 and 10μg mL−1 of free gossypol are extremely hazardous for in vitro bovine embryo development. Whether these deleterious effects take place in a similar fashion during in vivo embryo production remains to be investigated.


2004 ◽  
Vol 16 (4) ◽  
pp. 437 ◽  
Author(s):  
Yves Cognié ◽  
Nati Poulin ◽  
Yann Locatelli ◽  
Pascal Mermillod

Today, although not efficient enough to replace multiple ovulation and embryo transfer, in vitro embryo production for small ruminants is a platform for new reproductive technologies, such as embryo sexing, transgenesis and cloning. The in vitro embryo-production system developed for sheep and goats is more efficient now than 15 years ago, but could still be improved. Laparoscopic collection of oocytes in live animals treated with gonadotrophin indicates a promising future for the application of this technology to genetic improvement programmes. Oocyte maturation in defined medium with epidermal growth factor and cysteamine appears as efficient as oocyte maturation in follicular fluid-supplemented medium and allows future study of the effect of other factors involved in the cytoplasmic maturation of oocytes from these species. Further efforts have to be made to standardise the semen-capacitating process and to improve the quality and freezability of in-vitro-produced (IVP) embryos. The optimisation of IVP procedures for deer species has required the study of the seasonal variation of oocyte competence and the development of a specific methodology to allow the culture of embryos up to the blastocyst stage.


Animals ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 3248
Author(s):  
Morgan F. Orsolini ◽  
Stuart A. Meyers ◽  
Pouya Dini

As the use of assisted reproductive technologies (ART) and in vitro embryo production (IVP) expand in the equine industry, it has become necessary to further our understanding of semen physiology as it applies to overall fertility. This segment of our two-section review will focus on normal sperm parameters, beginning with development and extending through the basic morphology of mature spermatozoa, as well as common issues with male factor infertility in IVP. Ultimately, the relevance of sperm parameters to overall male factor fertility in equine IVP will be assessed.


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