The role of L-carnitine in bovine embryo metabolism. A review of the effect of supplementation with a metabolic modulator on in vitro embryo production

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in oocytes and early stages of pre-implantation embryos indicated a possible regulatory role for TH in these stages (unpublished data).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.

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In Vitro Bovine Embryo Production: The Role of FCS and Cysteamine on Cleavage Rate

Celal Bayar Üniversitesi Sağlık Bilimleri Enstitüsü Dergisi ◽

10.34087/cbusbed.624912 ◽

2020 ◽

Author(s):

Asiye İzem SANDAL

Keyword(s):

Bovine Embryo ◽

Cleavage Rate ◽

Embryo Production

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145 Inhibitory effects of gossypol on bovine in vitro embryo production

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab145 ◽

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

Keyword(s):

Embryo Development ◽

Free Gossypol ◽

Wilcoxon Test ◽

Free Form ◽

Bovine Embryo ◽

Sodium Pyruvate ◽

Embryo Production ◽

Parametric Data ◽

In Vitro Embryo Production

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 < 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 < 0.0001). As for the percentage of hatched blastocysts, the control (66.75%) had greater values compared with G5 (34.52%; P < 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.

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159 IN VITRO EMBRYO PRODUCTION FROM HOLSTEIN CALF OOCYTES RECOVERED BY LAPAROSCOPIC OVUM PICKUP

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab159 ◽

2016 ◽

Vol 28 (2) ◽

pp. 209

Author(s):

H. Baldassarre ◽

L. Currin ◽

L. Michalovic ◽

W. Glanzner ◽

K. Gutierrez ◽

...

Keyword(s):

Reproductive Organs ◽

Blastocyst Stage ◽

Developmental Competence ◽

Bovine Embryo ◽

Embryo Production ◽

Oocyte Competence ◽

Veterinary Practitioner ◽

Collection Tube ◽

In Vitro Embryo Production

Oocyte competence and reproductive biology in prepubertal heifer calves are not fully understood. Multiple publications have reported high oocyte yields recovered from calves aged 2–6 months old but low embryo development rates following in vitro embryo production. The objective of this study was to characterise the developmental competence of oocytes from young calves. We report herein the oocyte/embryo yields obtained from 6 Holstein heifer calves that were subjected to gonadotropin stimulation and laparoscopic ovum pick-up (LOPU) every 2 weeks, starting at 2 months of age and ending at 5 months of age. The LOPU was conducted under general anaesthesia with the animal lying in dorsal recumbency on a table with a 45-degree angle to facilitate the visualisation of reproductive organs. Briefly, looking through the laparoscope, the ovarian surface was exposed by pulling from the fimbria with an atraumatic grasping forceps. The follicle contents were aspirated using a 20 G needle mounted on a pipette, which was connected to a collection tube and a vacuum pump. Media and procedures for aspiration, in vitro maturation (IVM), IVF, and in vitro culture (IVC) were standard in use for commercial adult bovine embryo production. Because of the small number of animals and the multifactorial variables in play (age, number of previous treatments and aspirations, etc.), in this preliminary study we focused on the overall oocyte/embryo yield and the potential effects of LOPU on ovarian integrity. A total of 766 follicles were aspirated (avg. 17/calf per session) resulting in 625 cumulus-oocyte complexes (COC) recovered (avg. 14/calf per session; 82% recovery rate). A total of 457 (73%) COC were graded eligible for IVM, of which 353 cleaved (77%) and 109 (24%) reached a viable blastocyst stage at the end of IVC, of which 42 (38.5%) were graded as freezable. In balance, ~2 viable blastocysts/calf per session were produced. No adhesions or sequels were observed in the animals up to the last LOPU session, as well as 2 weeks after the last LOPU when the animals were evaluated by rectal palpation by an experienced OPU veterinary practitioner. Further studies will look into other aspects of oocyte developmental competence to better understand this biological process.

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222 COMPARISON OF COMMERCIAL IN VITRO EMBRYO PRODUCTION OF BRAHMAN DONORS UNDER BRAZILIAN v. PANAMANIAN MANAGEMENT

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab222 ◽

2011 ◽

Vol 23 (1) ◽

pp. 210

Author(s):

J. R. S. Chen ◽

L. F. Nasser ◽

L. Penteado ◽

M. Mendizabal ◽

A. C. Basso ◽

...

Keyword(s):

Large Scale ◽

Bos Taurus ◽

Bovine Embryo ◽

In Vitro Production ◽

Embryo Production ◽

Test Analysis ◽

Animal Biotechnology ◽

Follicular Aspiration ◽

In Vitro Embryo Production

Brazil is a leading country in the world in commercial use of in vitro-produced bovine embryos, with approximately 200 000 transfers per year (IETS; Thibier, 2009). This model of large-scale commercial in vitro bovine embryo production is now available for Panamanian producers. Because of the tropical environment in Panama, the most popular breed is the Brahman, a Zebu type of cattle that has been shown to have more follicles emerging per follicular wave than Bos taurus type of cattle and, consequently, that produce more oocytes per session of follicular aspiration. This characteristic, added to the embryo production results, permits such a biotechnology to be implemented on a commercial scale and incorporated into the reproduction management of a herd. A comparison of oocyte number and quality, cleavage, and embryo production was made using the same in vitro production system (InVitro Brazil, Mogi Mirim, São Paulo, Brazil) for Brahman donors, both in Brazil and in Panama. Data were compared using a z-test analysis (Table 1). The percentage of cleaved zygotes was greater (P < 0.001) with the Panamanian Brahman donors as compared with the Brazilian Brahman donors (73 v. 69%, respectively). However, the percentage of blastocysts/cleaved zygotes was greater (P < 0.01), indicating a higher blastocyst production rate from the Brazilian donors. No other differences were observed. Thus, in vitro embryo production with Brahman donors could be used as a tool to improve and spread superior genetics within a Panamanian herd and could also serve as a model for other Central American and Caribbean countries under similar management systems. Table 1.Panama and Brazil in vitro Brahman embryo production1 This work was supported by BORN Animal Biotechnology, Panama City, Panama.

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143 COMPARISON OF COMMERCIAL IN VITRO EMBRYO PRODUCTION AND PREGNANCY RATES OF BRAHMAN DONORS IN PANAMA VERSUS DOMINICAN REPUBLIC

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab143 ◽

2014 ◽

Vol 26 (1) ◽

pp. 185

Author(s):

A. Nagele ◽

E. Gomes ◽

A. Ruiz ◽

L. F. Nasser ◽

S. Feliu ◽

...

Keyword(s):

Dominican Republic ◽

Pregnancy Rate ◽

Large Scale ◽

Relative Number ◽

Management Systems ◽

Bovine Embryo ◽

In Vitro Production ◽

Embryo Production ◽

In Vitro Embryo Production

It has been previously demonstrated (IETS 2011) that Panama is applying the biotechnology of in vitro embryo production (IVP) to their bovine reproduction management systems. The present work demonstrates the ability to apply the IVP technology across 2 distant country borders. Herein, we demonstrate that a country (Dominican Republic; DR) that does not have a bovine IVP laboratory can take advantage of fresh bovine IVP embryos for transfer using distant IVP facilities in another country (Panama; ~1500 km away). The objective of this study was to demonstrate that a model system for large-scale commercial in vitro bovine embryo production for beef and dairy producers, that do not have IVP technology in their home country, could be developed producing comparable results. As the same laboratory provides IVP services to the both countries, a special sanitary protocol was developed in order to legalize the exchange of biological materials (oocytes or embryos). The data obtained in DR was compared to Panamanian client data because identical conditions were utilised for IVP. Cattle production systems were similar, as Brahman (a Zebu type of cattle) is the most popular breed in both countries. Oocytes were collected from 10 different herds in Panama and 4 different herds in DR. The oocytes were transported in an oocyte transporter in both instances. However, oocytes from DR were transported in InVitro Brasil™ maturation medium from 12 to 18 h and in Panama from 6 to 12 h before they were placed in a standard CO2 incubator. In both cases, the oocytes were matured for 24 h before fertilization with conventionally frozen Brahman semen in InVitro Brasil™ fertilization medium, followed by culture for up to 7 days in InVitro Brasil™ embryo culture medium. The embryos were transferred on Day 7, either in Panama or DR. They were transported by car in Panama and via airplane back to DR. A comparison of oocyte number and quality, cleavage, embryo production, and pregnancy rate, was made using the same in vitro production system for Brahman donors from September 2012 until May 2013. The difference between sites in the relative number of viable oocytes, relative number of cleaved oocytes among viable oocytes, relative number of embryos produced among cleaved oocytes, and relative number of embryos produced among viable oocytes was tested using Fisher's exact test. Pregnancy rate was analysed with chi-squared. We realise these results represent field data; however, we believe the present work is a significant step in demonstrating the potential for wide commercial-scale dissemination of IVP technology between distant countries. The number of embryos produced in Panama was slightly, but significantly, higher than those produced in DR; this is likely due to the larger number of donors and oocytes from the Panamanian herds. However, the pregnancy rate was higher in DR, likely due to the health status of DR recipients. These data illustrate that IVP using Brahman donors could be used as a tool to improve and spread superior genetics. Furthermore, this technique can serve as a model for other Central American and Caribbean countries under similar management systems. Table 1.Panama and the Dominican Republic in vitro Brahman embryo production and pregnancy (September 2012 through May 2013)

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The Role of Forskolin as a Lipolytic Stimulator during In Vitro Oocyte Maturation and the in Vitro Embryo Production of Livestock

Reproduction in Domestic Animals ◽

10.1111/rda.14021 ◽

2021 ◽

Author(s):

Sayed Haidar Abbas Raza ◽

Ayman H. Abd El‐Aziz ◽

Sameh A. Abdelnour ◽

Ahmed A. Easa ◽

Mahmoud Alagawany ◽

...

Keyword(s):

Oocyte Maturation ◽

Embryo Production ◽

In Vitro Embryo Production ◽

In Vitro Oocyte Maturation

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Detection of Brucella abortus during in vitro bovine embryo production

Bangladesh Journal of Livestock Research ◽

10.3329/bjlr.v27i1.55174 ◽

2021 ◽

pp. 105-112

Author(s):

A Akter ◽

GK Deb ◽

MFH Miraz ◽

MA Kabir ◽

SMJ Hossain ◽

...

Keyword(s):

Brucella Abortus ◽

Commercial Application ◽

Bovine Embryo ◽

Blastocyst Development ◽

Embryo Production ◽

Specific Pcr ◽

Contagious Diseases ◽

Biotechnological Approach ◽

In Vitro Embryo Production

The in vitro embryo production (IVP) technology has emerged as a potential biotechnological approach to multiply genetically high yielding dairy cows. Its commercial application is increasing in many developed and developing countries over the years. Bangladesh livestock Research Institute (BLRI) adopted in vitro embryo production protocol from bovine ovaries of slaughterhouse. However, the risks of transmission of contagious diseases like Brucella abortus with embryos are not evaluated so far. Considering these facts, the present experiments were conducted to evaluate the efficiency of in vitro embryo production protocol with slaughterhouse ovaries as well as risk of contamination of produced embryos with Brucella abortus. To identify sources of contamination of embryos with Brucella abortus (if any), the laboratory water, different media used in the IVP process, semen, and follicular fluids were evaluated for confirmation of the organisms. In addition, vaginal swabs were collected from 2 buffaloes aborted due to suspected Brucella abortus infection. Molecular test were used to detect Brucella abortus contamination. Brucella abortus specific PCR product was not detected on agarose gel electrophoresis. The efficiency of IVP measured by cleavage and blastocyst development rates were 75.5±2.7% and 16.6±3.9%, respectively. The present study inferred that the in vitro produce embryos are free from Brucella abortus infection. Bang. J. Livs. Res. Vol. 27 (1&2), 2020: P. 105-112

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102 IDENTIFICATION OF CONTRASTED PHENOTYPES IN THE BOVINE FROM REPEATED IN VIVO AND IN VITRO EMBRYO PRODUCTION FOLLOWING SUPEROVULATION

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab102 ◽

2008 ◽

Vol 20 (1) ◽

pp. 131 ◽

Cited By ~ 2

Author(s):

C. Guyader-Joly ◽

S. Ponchon ◽

C. Gonzalez ◽

B. Marquant-Le Guienne ◽

L. Clément ◽

...

Keyword(s):

Developmental Competence ◽

Marker Genes ◽

Bovine Embryo ◽

Embryo Production ◽

Cell Monolayers ◽

In Vitro Embryo Production ◽

Vitro Development

This study was initiated to evaluate maternal influence on in vivo and in vitro bovine embryo production and identify animals with contrasted phenotypes for reproductive parameters. Nine Montbéliard cows raised on the same farm and with various genetic origins were included in the study. In vivo-derived embryos were collected nonsurgically from superovulated cows on day 7 after AI (34 collections). Immature oocytes were collected by ovum pickup from the same (superovulated) cows (36 sessions) then matured, fertilized (day 0) with the same bull, and cultured in vitro until day 7 on Vero cell monolayers in B2 medium. Grade 1 to 3 in vivo and grade 1 and 2 in vitro produced embryos deemed viable according to IETS criteria. The mean numbers of blastocysts and viable blastocysts per session per cow were, respectively, 8.3 ± 5.5 and 4.8 ± 3.6 in the in vivo system and 2.5 ± 2.6 and 1.8 ± 2.2 in the in vitro system. Individual cow data of in vivo and in vitro embryo production were analyzed by ANOVA (GLM program in SAS; SAS Institute Inc., Cary, NC, USA). Results are presented in Table 1: mean ± SD. Quantity and quality of produced embryos varied significantly among females, and production in vivo and in vitro was not systematically related. Contrasted phenotypes were identified according to their viable blastocyst rates in both systems (in vivo: no viable/recovered; in vitro: no viable/inseminated). Two females presented a relatively high percentage of viable blastocysts in both systems (over 30% in vitro and over 70% in vivo, Table 1). On the contrary, 2 females showed low percentages of blastocysts in the 2 systems (<10% in vitro and <50% in vivo). For most other females, the percentage of in vivo-produced blastocysts was relatively high (>50%), but in vitro development rates were low. Only one female (C3) presented the inverse situation. Oocytes collected from animals with contrasted phenotypes will be analysed for gene expression to identify marker genes associated with oocyte developmental competence. Table 1. This study was conducted with financial support of ‘Genanimal’ – French Ministry of Research (#03P409) and Apis-Gene.

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

Revista Colombiana de Ciencias Pecuarias ◽

10.17533/udea.rccp.v32n2a02 ◽

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 ◽

...

Keyword(s):

Genetic Diversity ◽

Microsatellite Markers ◽

Assisted Reproductive Technologies ◽

Reproductive Technologies ◽

Bovine Embryo ◽

Embryo Production ◽

Low Genetic Diversity ◽

In Vitro Embryo Production ◽

Population Interactions

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.

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