RNA sequencing and gene co-expression network of in vitro matured oocytes and blastocysts of buffalo.

In reproductive technologies, uncovering the molecular aspects of oocyte and embryo competence under different conditions is crucial for refining protocols and enhancing efficiency. RNA-seq generates high-throughput data and provides transcriptomes that can undergo additional computational analyses. This study presented the transcriptomic profiles of in vitro matured oocytes and blastocysts produced in vitro from buffalo crossbred (Bubalus bubalis), coupled with gene co-expression and module preservation analysis. Cumulus Oophorus Complexes, obtained from slaughterhouse-derived ovaries, were subjected to in vitro maturation to yield metaphase II oocytes (616) or followed in vitro fertilization and culture to yield blastocysts for sequencing (526). Oocyte maturation (72%, ±3.34 sd) and embryo development (21.3%, ±4.18 sd) rates were obtained from three in vitro embryo production routines following standard protocols. Sequencing of 410 metaphase II oocytes and 70 hatched blastocysts (grade 1 and 2) identified a total of 13,976 genes, with 62% being ubiquitously expressed (8,649). Among them, the differentially expressed genes (4,153) and the strongly variable genes with the higher expression (fold-change above 11) were highlighted in oocytes (BMP15, UCHL1, WEE1, NLRPs, KPNA7, ZP2, and ZP4) and blastocysts (APOA1, KRT18, ANXA2, S100A14, SLC34A2, PRSS8 and ANXA2) as representative indicators of molecular quality. Additionally, genes exclusively found in oocytes (224) and blastocysts (2,200) with specific biological functions were identified. Gene co-expression network and module preservation analysis revealed strong preservation of functional modules related to exosome components, steroid metabolism, cell proliferation, and morphogenesis. However, cell cycle and amino acid transport modules exhibited weak preservation, which may reflect differences in embryo development kinetics and the activation of cell signaling pathways between buffalo and bovine. This comprehensive transcriptomic profile serves as a valuable resource for assessing the molecular quality of buffalo oocytes and embryos in future in vitro embryo production assays.

Use of platelet-rich plasma on in vitro maturation during bovine embryo production.

One of the crucial aspects to be considered for successful in vitro production (IVP) of embryos is the composition of the various media used throughout the stages of this reproductive biotechnology. The cell culture media employed should fulfill the metabolic requirements of both gametes during oocyte maturation and sperm development, as well as the embryo during its initial cell divisions. Most IVP protocols incorporate blood serum into the media composition as a source of hormones, proteins, growth factors, and nutrients. Numerous studies have suggested Platelet-Rich Plasma (PRP) as a substitute for fetal sera in cell culture, particularly for stem cells. Therefore, the objective of this study is to assess the potential use of PRP as a replacement for fetal bovine serum (FBS) during oocyte maturation for in vitro production of bovine embryos. During in vitro maturation (IVM), cumulus-oocyte complexes (COCs) were allocated into the following experimental groups: Group G1 (IVM medium with 5% PRP); Group G2 (MIV medium with 5% PRP and 5% SFB); Group G3 (MIV medium with 5% SFB); and Group G4 (MIV medium without either PRP or SFB). Subsequently, the cumulus-oocyte complexes were fertilized with semen from a single bull, and the resulting zygotes were cultured for seven days. Cleavage and blastocyst formation rates were assessed on days 2 and 7 of embryonic development, respectively. The quality of matured COCs was also evaluated by analyzing the gene expression of HSP70, an important protein associated with cellular stress. The results demonstrated that there were no significant differences among the experimental groups in terms of embryo production rates, both in the initial cleavage stages and blastocyst formation (except for the G4 group, which exhibited a lower blastocyst formation rate on D7, as expected). This indicates that PRP could be a cost-effective alternative to SFB in the IVP of embryos.

Sperm chromatin protamination influences embryo development in unsexed and sexed bull semen.

Sex selection through sperm sorting offers advantages in regards selection pressure in high-producing livestock. However, the sex-sorting process results in sperm membrane and DNA damage that ultimately decrease fertility. We hypothesized that given the role of protamines in DNA packaging, protamine deficiency could account, at least partially, for the DNA damage observed following sperm sex sorting. To test this, we compared protamine status between unsexed and sexed spermatozoa from two bulls using the fluorochrome chromomycin A3 (CMA3) and flow cytometry. Then, we assessed embryo development following in vitro fertilization (IVF) using the same sperm treatments. Overall, sperm protamination was not different between sexed and unsexed semen. However, one of the two bulls displayed higher rates of protamine deficiency for both unsexed and sexed semen (P < 0.05). Moreover, unsexed semen from this bull yielded lower blastocyst (P < 0.05) and blastocyst hatching rates than unsexed sperm from the other bull. CMA3-positive staining was negatively correlated with cleavage (R2 85.1, P = 0.003) and blastocyst hatching (R2 87.6, P = 0.006) rates in unsexed semen. In conclusion, while the sex-sorting process had no effect on sperm protamine content, we observed a bull effect for sperm protamination, which correlated to embryo development rates following IVF.

In vitro embryo production in buffaloes: from the laboratory to the farm.

Transvaginal follicular aspiration technique together with in vitro embryo production are the biotechnological alternatives currently available to support genetic improvement breeding programs in buffalo species. However, aspects related to animal management, lack of knowledge of the metabolic needs and biochemical peculiarities of gametes and embryos, as well as the reproductive physiology characteristics have hampered progress in the results. Despite the low availability of good quality oocytes collected after OPU in donors as a physiological characteristic of buffalo species, high rates of oocyte maturation, modest embryo cleavage, blastocyst production and pregnancy rates after transvaginal embryo transfer in recipients could be obtained in buffalo in vitro embryo production programs. The results of implementing an in vitro embryo production program in buffaloes in the northern region of Pará state, Brazil, and results published by other groups demonstrate the feasibility of implementing this biotechnology in the routine of breeding programs. Nevertheless, in order to achieve better and consistent results, it is necessary to deepen the knowledge on the peculiarities of reproductive biology in this specie. Selection of donor animals based on ovarian size and ovarian follicular reserve and on the rate of blastocyst production is presented as an effective alternative to increase the efficiency of the in vitro embryo production technique applied to the buffalo species.

Contributions of RNA-seq to improve in vitro embryo production (IVP).

In Vitro Embryo Production (IVP) is widely used to improve the reproductive efficiency of livestock animals, however increasing the embryo development rates and pregnancy outcomes is still a challenge for some species. Thus, the lack of biological knowledge hinders developing specie-specific IVP protocols. Therefore, the contributions of RNA-seq to generate relevant biological knowledge and improve the efficiency of IVP in livestock animals are reviewed herein.

Contributions of RNA-seq to improve in vitro embryo production (IVP)

In Vitro Embryo Production (IVP) is widely used to improve the reproductive efficiency of livestock animals, however increasing the embryo development rates and pregnancy outcomes is still a challenge for some species. Thus, the lack of biological knowledge hinders developing specie-specific IVP protocols. Therefore, the contributions of RNA-seq to generate relevant biological knowledge and improve the efficiency of IVP in livestock animals are reviewed herein.

In vitro embryo production in buffaloes: from the laboratory to the farm

Transvaginal follicular aspiration technique together with in vitro embryo production are the biotechnological alternatives currently available to support genetic improvement breeding programs in buffalo species. However, aspects related to animal management, lack of knowledge of the metabolic needs and biochemical peculiarities of gametes and embryos, as well as the reproductive physiology characteristics have hampered progress in the results. Despite the low availability of good quality oocytes collected after OPU in donors as a physiological characteristic of buffalo species, high rates of oocyte maturation, modest embryo cleavage, blastocyst production and pregnancy rates after transvaginal embryo transfer in recipients could be obtained in buffalo in vitro embryo production programs. The results of implementing an in vitro embryo production program in buffaloes in the northern region of Pará state, Brazil, and results published by other groups demonstrate the feasibility of implementing this biotechnology in the routine of breeding programs. Nevertheless, in order to achieve better and consistent results, it is necessary to deepen the knowledge on the peculiarities of reproductive biology in this specie. Selection of donor animals based on ovarian size and ovarian follicular reserve and on the rate of blastocyst production is presented as an effective alternative to increase the efficiency of the in vitro embryo production technique applied to the buffalo species.

Efeito de diferentes osmolaridades do diluidor Tris-citrato na congelação com etilenoglicol ou glicerol de espermatozoides epididimários de gato doméstico (Felis catus) / Effect of different osmolarities of Tris-citrate extender on freezing epididymal sperm from domestic cat (Felis catus)with ethyleneglycol or glycerol

Objetivou-se com o estudo investigar o efeito de diferentes osmolaridades do diluidor Triscitrato em espermatozóides epididimários de gatos domésticos (Feliscatus) e a congelação com glicerol ou etilenoglicol. Foram realizados dois experimentos, com 10 gatos. No Experimento 1, avaliou-se a manutenção dos parâmetros espermáticos em diluidor tris-citrato com osmolaridades 275, 325, 375, 425, 475 e 525mOsm, nos tempos (T0= 0, T1= 30 e T2= 60 min). No Experimento 2 a congelação foi realizada utilizando as osmolaridades 325 e 375 do glicerol a 4% ou etilenoglicol a 3%, 6%. Dentre as osmolaridades, quanto à motilidade a 325 mOsm não diferiu estatisticamente com o fluido epidídimal (controle) nos três tempos e a 375 mOsm no T0 e T1, e ambas não apresentaram diferenças estatísticas entre si e entre os tempos em todos os parâmetros espermáticos. O uso de 4% de glicerol em diluidor com 375 mOsm foi superior, apresentando motilidade de 25% ± 6, vigor 4, integridade de membrana plasmática de 48% ± 9, sem diferenças estatísticas com o resfriamento e na morfologia não foram encontradas diferenças estatísticas entre as duas osmolaridades. Portanto, o Tris-citrato com 325 e 375 mOsm entre as osmolaridades testadas e pós congelação com 375 mOsm e glicerol 4% manteve os parâmetros espermáticos.

The aim of this study was to investigate the effect of different osmotic potentials of Tris-citrate extender in epididymal spermatozoa of domestic cats (Felis catus), frozen with glycerol or ethyleneglycol. Two experiments were carried out, with ten cats. In the first experiment, the influence of extender with the osmolarityof 275, 325, 375, 425, 475 and 525 mOsm on sperm parameters were evaluated. In the second experiment, slow freezing was performed using glycerol at 4% or ethyleneglycolat 3% and 6% added to extender with 325and 375 mOsm. Among the osmolarities, the motilityat 325 mOsm did not differ statistically with epididymal fluid (control) at all times evaluated and at 375 mOsmat T0 and T1, and both showed no statistical differences between each other and between the times in all sperm parameters. Glycerol 4% added to extender with 475 mOsm was superior, presenting motilityof 25% ± 6, vigor 4, plasma membrane integrity of 48% ± 9, without statistical differences with cooling and in morphology, no statistical differences were found between the two osmolarities. Therefore, 325 and 375 mOsm Triscitrate between the osmolarities tested and after freezing with 375 mOsm and 4%, glycerol maintained the sperm parameters.

Contributions of RNA-seq to improve in vitro embryo production (IVP)

In Vitro Embryo Production (IVP) is widely used to improve the reproductive efficiency of livestock animals, however increasing the embryo development rates and pregnancy outcomes is still a challenge for some species. Thus, the lack of biological knowledge hinders developing specie-specific IVP protocols. Therefore, the contributions of RNA-seq to generate relevant biological knowledge and improve the efficiency of IVP in livestock animals are reviewed herein.(AU)

In vitro embryo production in buffaloes: from the laboratory to the farm

Transvaginal follicular aspiration technique together with in vitro embryo production are the biotechnological alternatives currently available to support genetic improvement breeding programs in buffalo species. However, aspects related to animal management, lack of knowledge of the metabolic needs and biochemical peculiarities of gametes and embryos, as well as the reproductive physiology characteristics have hampered progress in the results. Despite the low availability of good quality oocytes collected after OPU in donors as a physiological characteristic of buffalo species, high rates of oocyte maturation, modest embryo cleavage, blastocyst production and pregnancy rates after transvaginal embryo transfer in recipients could be obtained in buffalo in vitro embryo production programs. The results of implementing an in vitro embryo production program in buffaloes in the northern region of Pará state, Brazil, and results published by other groups demonstrate the feasibility of implementing this biotechnology in the routine of breeding programs. Nevertheless, in order to achieve better and consistent results, it is necessary to deepen the knowledge on the peculiarities of reproductive biology in this specie. Selection of donor animals based on ovarian size and ovarian follicular reserve and on the rate of blastocyst production is presented as an effective alternative to increase the efficiency of the in vitro embryo production technique applied to the buffalo species.(AU)

Efeito de diferentes osmolaridades do diluidor Tris-citrato na congelação com etilenoglicol ou glicerol de espermatozoides epididimários de gato doméstico (Felis catus) / Effect of different osmolarities of Tris-citrate extender on freezing epididymal sperm from domestic cat (Felis catus)with ethyleneglycol or glycerol

Objetivou-se com o estudo investigar o efeito de diferentes osmolaridades do diluidor Triscitrato em espermatozóides epididimários de gatos domésticos (Feliscatus) e a congelação com glicerol ou etilenoglicol. Foram realizados dois experimentos, com 10 gatos. No Experimento 1, avaliou-se a manutenção dos parâmetros espermáticos em diluidor tris-citrato com osmolaridades 275, 325, 375, 425, 475 e 525mOsm, nos tempos (T0= 0, T1= 30 e T2= 60 min). No Experimento 2 a congelação foi realizada utilizando as osmolaridades 325 e 375 do glicerol a 4% ou etilenoglicol a 3%, 6%. Dentre as osmolaridades, quanto à motilidade a 325 mOsm não diferiu estatisticamente com o fluido epidídimal (controle) nos três tempos e a 375 mOsm no T0 e T1, e ambas não apresentaram diferenças estatísticas entre si e entre os tempos em todos os parâmetros espermáticos. O uso de 4% de glicerol em diluidor com 375 mOsm foi superior, apresentando motilidade de 25% ± 6, vigor 4, integridade de membrana plasmática de 48% ± 9, sem diferenças estatísticas com o resfriamento e na morfologia não foram encontradas diferenças estatísticas entre as duas osmolaridades. Portanto, o Tris-citrato com 325 e 375 mOsm entre as osmolaridades testadas e pós congelação com 375 mOsm e glicerol 4% manteve os parâmetros espermáticos.(AU)

The aim of this study was to investigate the effect of different osmotic potentials of Tris-citrate extender in epididymal spermatozoa of domestic cats (Felis catus), frozen with glycerol or ethyleneglycol. Two experiments were carried out, with ten cats. In the first experiment, the influence of extender with the osmolarityof 275, 325, 375, 425, 475 and 525 mOsm on sperm parameters were evaluated. In the second experiment, slow freezing was performed using glycerol at 4% or ethyleneglycolat 3% and 6% added to extender with 325and 375 mOsm. Among the osmolarities, the motilityat 325 mOsm did not differ statistically with epididymal fluid (control) at all times evaluated and at 375 mOsmat T0 and T1, and both showed no statistical differences between each other and between the times in all sperm parameters. Glycerol 4% added to extender with 475 mOsm was superior, presenting motilityof 25% ± 6, vigor 4, plasma membrane integrity of 48% ± 9, without statistical differences with cooling and in morphology, no statistical differences were found between the two osmolarities. Therefore, 325 and 375 mOsm Triscitrate between the osmolarities tested and after freezing with 375 mOsm and 4%, glycerol maintained the sperm parameters.(AU)

Desafio da Pecuária na Amazônia frente ao novo código florestal brasileiro / Challenge of Livestock in the Amazon in front of the new Brazilian forest law

O rebanho bovino brasileiro no período de 1975 a 2017 passou de 101.673.753 para 171.858.168 de animais, o que representa um crescimento de 69%, sendo que 77% desse crescimento ocorreu na Amazônia, onde no mesmo período, o rebanho cresceu 1.066%. Mas, a derrubada da floresta cresceu na mesma proporção colocando a região no centro das discussões nacionais e internacionais em relação ao meio ambiente. Em 2012 foi aprovado o novo código florestal o qual determina que na Amazônia deve ser preservado 80% da área da propriedade como reserva legal (RL), restando 20% a ser explorado economicamente. Apesar da questão ambiental, é inegável que a pecuária colocou a região dentro do contexto econômico nacional. Portanto, o desafio é: é possível manter o desenvolvimento da pecuária na Amazônia de forma sustentável, dentro da concepção da nova lei? É dentro desse contexto que abordaremos de forma superficial essa grande questão, na certeza de que para respondê-la precisará de um profundo debate pela sociedade.

The Brazilian cattle herd in between 1975 and 2017 rose from 101,673,753 to 171,858,168 of animals, representing a growth of 69%, of which 77% of this growth occurred in the Amazon, where in the same period the herd grew 1.066%. However, the deforestation has grown at the same rate, putting the region at the center of national and international environmental discussions. In 2012 it was approved the new forest law that states that 80% of the property area must be kept as legal reserve (RL), whereas only 20% remaining economically exploitable. Despite the environmental issue, it is undeniable that livestock farming has placed the region within the national economic context. So the challenge is: Is it possible to sustainably maintain the livestock development in the Amazon region? It is within this context that we will superficially address this great question, in the certainty that in order to answer it, it will require a deep debate by society.

Desafio da Pecuária na Amazônia frente ao novo código florestal brasileiro / Challenge of Livestock in the Amazon in front of the new Brazilian forest law

O rebanho bovino brasileiro no período de 1975 a 2017 passou de 101.673.753 para 171.858.168 de animais, o que representa um crescimento de 69%, sendo que 77% desse crescimento ocorreu na Amazônia, onde no mesmo período, o rebanho cresceu 1.066%. Mas, a derrubada da floresta cresceu na mesma proporção colocando a região no centro das discussões nacionais e internacionais em relação ao meio ambiente. Em 2012 foi aprovado o novo código florestal o qual determina que na Amazônia deve ser preservado 80% da área da propriedade como reserva legal (RL), restando 20% a ser explorado economicamente. Apesar da questão ambiental, é inegável que a pecuária colocou a região dentro do contexto econômico nacional. Portanto, o desafio é: é possível manter o desenvolvimento da pecuária na Amazônia de forma sustentável, dentro da concepção da nova lei? É dentro desse contexto que abordaremos de forma superficial essa grande questão, na certeza de que para respondê-la precisará de um profundo debate pela sociedade.(AU)

The Brazilian cattle herd in between 1975 and 2017 rose from 101,673,753 to 171,858,168 of animals, representing a growth of 69%, of which 77% of this growth occurred in the Amazon, where in the same period the herd grew 1.066%. However, the deforestation has grown at the same rate, putting the region at the center of national and international environmental discussions. In 2012 it was approved the new forest law that states that 80% of the property area must be kept as legal reserve (RL), whereas only 20% remaining economically exploitable. Despite the environmental issue, it is undeniable that livestock farming has placed the region within the national economic context. So the challenge is: Is it possible to sustainably maintain the livestock development in the Amazon region? It is within this context that we will superficially address this great question, in the certainty that in order to answer it, it will require a deep debate by society.(AU)

Produção in vitro de embrião (PIVE) na espécie bubalina / In vitro embryo production of water buffalo (Bubalus bubalis)

Nos últimos anos a biotécnica de produção in vitro de embrião (PIVE) em bubalino passou porimportantes modificações, possibilitando a sua aplicação ao nível comercial. Entretanto, a taxa de produção deembrião por doadora ainda é muito baixa, refletindo diretamente no custo da prenhez, o qual é cerca de 3-4 vezesmais elevado que aquele observado em bovino zebuíno. Essa baixa taxa de embrião ocorre em função de váriosfatores, dentre os mais importantes podemos citar os fatores intrínsecos relacionados aos animais, como a baixaquantidade e qualidade dos oócitos obtidos por doadora; mas também a fatores extrínsecos, como a falta demeios de cultivo específicos para o oócito/embrião da espécie, as deficiências nas condições de manejo(nutricional e sanitário). Portanto, para a aplicação comercial mais abrangente, é necessário baixar o custo daprenhez, bem como, disponibilizar animais de comprovada produtividade (teste de progênie) para utilização emprogramas de PIVE e que do ponto de vista custo / benefício compense a utilização dessa técnica.

In recent years, the biotechnology of in vitro embryo production (IVP) in buffalo has undergone a greatimprovement, allowing its application at the commercial level. However, the rate of embryo production perdonor is still very low, reflecting directly on the cost of pregnancy that is about 3-4 times higher than zebu cattle.This low embryo rate occurs due to several factors. Among the most important are the intrinsic factors related tothe animals such as the low quantity and quality of the oocytes obtained by the donor, but also to extrinsicfactors such as the lack of specific culture media for the oocyte/embryo, and deficiencies in the managementconditions, that could be nutritional or sanitary. Therefore, for the expansion in commercial application, it isnecessary to reduce the cost of pregnancy, as well as to provide animals of proven productivity (progeny test) foruse in PIVE programs and that from the cost / benefit point of view compensate the use of a high cost techniquesuch as PIVE.

Produção in vitro de embrião (PIVE) na espécie bubalina / In vitro embryo production of water buffalo (Bubalus bubalis)

Nos últimos anos a biotécnica de produção in vitro de embrião (PIVE) em bubalino passou porimportantes modificações, possibilitando a sua aplicação ao nível comercial. Entretanto, a taxa de produção deembrião por doadora ainda é muito baixa, refletindo diretamente no custo da prenhez, o qual é cerca de 3-4 vezesmais elevado que aquele observado em bovino zebuíno. Essa baixa taxa de embrião ocorre em função de váriosfatores, dentre os mais importantes podemos citar os fatores intrínsecos relacionados aos animais, como a baixaquantidade e qualidade dos oócitos obtidos por doadora; mas também a fatores extrínsecos, como a falta demeios de cultivo específicos para o oócito/embrião da espécie, as deficiências nas condições de manejo(nutricional e sanitário). Portanto, para a aplicação comercial mais abrangente, é necessário baixar o custo daprenhez, bem como, disponibilizar animais de comprovada produtividade (teste de progênie) para utilização emprogramas de PIVE e que do ponto de vista custo / benefício compense a utilização dessa técnica.(AU)

In recent years, the biotechnology of in vitro embryo production (IVP) in buffalo has undergone a greatimprovement, allowing its application at the commercial level. However, the rate of embryo production perdonor is still very low, reflecting directly on the cost of pregnancy that is about 3-4 times higher than zebu cattle.This low embryo rate occurs due to several factors. Among the most important are the intrinsic factors related tothe animals such as the low quantity and quality of the oocytes obtained by the donor, but also to extrinsicfactors such as the lack of specific culture media for the oocyte/embryo, and deficiencies in the managementconditions, that could be nutritional or sanitary. Therefore, for the expansion in commercial application, it isnecessary to reduce the cost of pregnancy, as well as to provide animals of proven productivity (progeny test) foruse in PIVE programs and that from the cost / benefit point of view compensate the use of a high cost techniquesuch as PIVE.(AU)

Supplementation of bovine embryo culture medium with L-arginine improves embryo quality via nitric oxide production.

Nitric oxide (NO) is a cell-signaling molecule that regulates a variety of molecular pathways. We investigated the role of NO during preimplantation embryonic development by blocking its production with an inhibitor or supplementing in vitro bovine embryo cultures with its natural precursor, L-arginine, over different periods. Endpoints evaluated included blastocyst rates, development kinetics, and embryo quality. Supplementation with the NO synthase inhibitor N-Nitro-L-arginine-methyl ester (L-NAME) from Days 1 to 8 of culture decreased blastocyst (P < 0.05) and hatching (P < 0.05) rates. When added from Days 1 to 8, 50 mM L-arginine decreased blastocyst rates (P < 0.001); in contrast, when added from Days 5 to 8, 1 mM L-arginine improved embryo hatching rates (P < 0.05) and quality (P < 0.05) as well as increased POU5F1 gene expression (P < 0.05) as compared to the untreated control. Moreover, NO levels in the medium during this culture period positively correlated with the increased embryo hatching rates and quality (P < 0.05). These data suggest exerts its positive effects during the transition from morula to blastocyst stage, and that supplementing the embryo culture medium with L-arginine favors preimplantation development of bovine embryos.