An Update on Semen Physiology, Technologies, and Selection Techniques for the Advancement of In Vitro Equine Embryo Production: Section II

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.

Download Full-text

An Update on Semen Physiology, Technologies, and Selection Techniques for the Advancement of In Vitro Equine Embryo Production: Section I

Animals ◽

10.3390/ani11113248 ◽

2021 ◽

Vol 11 (11) ◽

pp. 3248

Author(s):

Morgan F. Orsolini ◽

Stuart A. Meyers ◽

Pouya Dini

Keyword(s):

Assisted Reproductive Technologies ◽

Reproductive Technologies ◽

Sperm Parameters ◽

Male Factor Infertility ◽

Male Factor ◽

Embryo Production ◽

Normal Sperm ◽

Production Section ◽

Equine Industry

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.

Download Full-text

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.

Download Full-text

Selection of stallions for in vitro embryo production by ICSI in a commercial program

Journal of Equine Veterinary Science ◽

10.1016/j.jevs.2012.05.031 ◽

2012 ◽

Vol 32 (7) ◽

pp. 409 ◽

Cited By ~ 6

Author(s):

C. Herrera ◽

P. Dufourq ◽

M. Freije ◽

I. Morikawa ◽

J.E. Centeno ◽

...

Keyword(s):

Embryo Production ◽

Commercial Program ◽

In Vitro Embryo Production ◽

Selection Of

Download Full-text

34 Reproductive management of beef females in tropical and sub-tropical environments

Journal of Animal Science ◽

10.1093/jas/skz258.074 ◽

2019 ◽

Vol 97 (Supplement_3) ◽

pp. 37-37

Author(s):

Nicola Oosthuizen ◽

Pedro Levy Piza Fontes ◽

G Cliff Lamb

Keyword(s):

Assisted Reproductive Technologies ◽

Bos Taurus ◽

Management Strategies ◽

Bos Indicus ◽

The United States ◽

Reproductive Technologies ◽

Pregnancy Rates ◽

Embryo Production ◽

Reproductive Management

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.

Download Full-text

245 OOCYTE RECOVERY BY OVUM PICK UP AND EMBRYO PRODUCTION IN MURRAH AND NILI-RAVI BUFFALOES (BUBALUS BUBALIS) IMPORTED IN CHINA

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab245 ◽

2005 ◽

Vol 17 (2) ◽

pp. 273 ◽

Cited By ~ 5

Author(s):

Y. Huang ◽

X. Zhang ◽

B. Gasparrini ◽

G.A. Presicce

Keyword(s):

Essential Amino Acids ◽

Reproductive Technologies ◽

Meat Production ◽

Embryo Production ◽

Vitro Fertilization ◽

Preliminary Results ◽

Swamp Buffaloes ◽

In Vitro Embryo Production ◽

Humidified Air

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.

Download Full-text

123 LONG-TERM PRODUCTION OF IN VITRO-MATURED PORCINE OOCYTES AND EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab123 ◽

2009 ◽

Vol 21 (1) ◽

pp. 161

Author(s):

A. M. Paprocki ◽

C. M. Syverson ◽

R. W. Koppang ◽

J. R. Dobrinsky

Keyword(s):

Assisted Reproductive Technologies ◽

Genetic Material ◽

Reproductive Technologies ◽

Scientific Data ◽

Developmental Competence ◽

Embryo Production ◽

Pump System ◽

Developmental Potential ◽

Parthenogenetic Embryos

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.

Download Full-text

State-of-the-art production, conservation and transfer of in-vitro-produced embryos in small ruminants

Reproduction Fertility and Development ◽

10.1071/rd04029 ◽

2004 ◽

Vol 16 (4) ◽

pp. 437 ◽

Cited By ~ 57

Author(s):

Yves Cognié ◽

Nati Poulin ◽

Yann Locatelli ◽

Pascal Mermillod

Keyword(s):

Oocyte Maturation ◽

Small Ruminants ◽

Reproductive Technologies ◽

Defined Medium ◽

Blastocyst Stage ◽

Embryo Production ◽

Oocyte Competence ◽

Epidermal Growth ◽

In Vitro Embryo Production

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.

Download Full-text

Update on advanced semen-processing technologies and their application for in vitro embryo production in horses

Reproduction Fertility and Development ◽

10.1071/rd19301 ◽

2019 ◽

Vol 31 (12) ◽

pp. 1771

Author(s):

Lee H. Morris ◽

Lisa J. Maclellan

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Reproductive Technologies ◽

Breeding Program ◽

Embryo Production ◽

Processing Technologies ◽

Advanced Technologies ◽

The Status ◽

In Vitro Embryo Production ◽

Semen Processing

The increased commercialisation of intracytoplasmic sperm injection (ICSI) in horses creates more opportunities to incorporate advanced reproductive technologies, such as sex-sorted, refrozen and lyophilised spermatozoa, into a breeding program. This paper reviews the status of these semen-handling technologies in light of their use in equine ICSI programs. Pregnancies have been achieved from each of these advanced technologies when combined with ICSI in horses, but refinements in the semen-handling processes underpinning these technologies are currently being explored to produce more reliable and practical improvements in the results from equine ICSI.

Download Full-text

83 Pregnancy losses after transfer of bovine embryos produced by assisted reproductive technologies

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab83 ◽

2019 ◽

Vol 31 (1) ◽

pp. 167

Author(s):

L. F. Feres ◽

M. P. Palhao ◽

L. G. B. Siqueira ◽

J. H. M. Viana

Keyword(s):

Risk Factors ◽

Developmental Stage ◽

Corpus Luteum ◽

Pregnancy Loss ◽

Assisted Reproductive Technologies ◽

Reproductive Technologies ◽

Quality Score ◽

Pregnancy Rates ◽

Embryo Production

The aim of this study was to evaluate risk factors for pregnancy loss in embryo recipients. We evaluated data from 31,792 embryo transfers (ET), including 859 embryos derived in vivo (IVD), 28,814 produced in vitro (IVP), and 2,119 produced by somatic cell nuclear transfer (SCNT), recorded from 2008 to 2012 on a single commercial farm. Embryos were produced by different in vitro embryo production laboratories, but ET was performed by the same team and within the same herd. Recipients were checked for pregnancy (PR) by ultrasonography 23 days after ET and re-examined on Days 60, 90, and 120 of gestation to identify pregnancy losses (PL). Data were analysed for the main effect of embryo production technique upon PL. For IVD and IVP embryos only, additional risk factors were included in the statistical model, including cryopreservation, type of semen (conventional, sex-sorted, reverse sex-sorted), embryo developmental stage, recipient corpus luteum quality score, use of natural oestrus or synchronization protocol, embryo-recipient synchrony, embryo genetics (taurine v. zebu, dairy v. beef), season (summer v. winter), and fetal sex. Data were analysed using the GLIMMIX procedure of the SAS Software (v 9.3, SAS Institute Inc., Cary, NC, USA). Overall PR was 42.1% (13,395 of 31,792) and PL between days 30 and 60, 60 and 90, and >90 were 8.7, 5.3, and 3.5%, respectively (cumulative PL=17.4%). The SCNT embryos had lower PR compared to IVP and IVD embryos (36.6, 42.5, and 44.2%, respectively; P<0.001) as well as a greater PL in all periods (30-60=45.2, 6.4, and 8.2%; 60-90=3.5, 1.4, and 1.8%; and >90=21.4, 6.3, and 7.1%, respectively; P<0.0001). The PR was similar between fresh and frozen embryos, but lower for vitrified ones (42.9, 43.1, and 39.1%, respectively, P<0.05). Slow freezing resulted in a greater total PL compared to fresh embryos (18.0v. 13.9%, respectively; P<0.05). We observed an interaction between embryo genetics and season. The taurine dairy subgroup had the lowest PR during summer (37.7v. >41.7% in all other groups; P<0.01), but within this group PR increased during winter (37.7v. 44.6%, P<0.05). The PL was greater for taurine than zebu during summer (17.8v. 14.0%; P<0.001), but did not differ during winter (P>0.05). The type of semen, embryo developmental stage, corpus luteum quality score, and embryo recipient synchrony affected pregnancy rates (P<0.05) but not pregnancy losses (P>0.05). Interestingly, the use of ovulation synchronization protocols resulted in similar pregnancy rates but greater pregnancy losses compared to natural oestrus (41.8v. 43.2%, P>0.05; and 15.9v. 22.1%, P<0.05, respectively). Pregnancy loss after 60 days was similar between female and male fetuses (5.0v. 4.9%, respectively; P>0.05). In summary, the assisted technology used to produce and freeze embryos was the main cause of variation in pregnancy losses, which were also affected by the interplay of season by genetic background and by the use of synchronization protocols. Conversely, factors known to affect pregnancy rates, such as embryo developmental stage, corpus luteum quality, and embryo-recipient synchrony, had no effect upon pregnancy loss. Research was supported by Mineirembryo, CAPES, and FAPDF.

Download Full-text

Year-Long Phenotypical Study of Calves Derived From Different Assisted-Reproduction Technologies

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.739041 ◽

2022 ◽

Vol 8 ◽

Author(s):

Jordana S. Lopes ◽

Cristina Soriano-Úbeda ◽

Evelyne París-Oller ◽

Sergio Navarro-Serna ◽

Analuce Canha-Gouveia ◽

...

Keyword(s):

Embryo Culture ◽

Growth And Development ◽

Assisted Reproductive Technologies ◽

Culture Media ◽

Reproductive Technologies ◽

Embryo Production ◽

Cattle Industry ◽

Entire Experiment

Assisted reproductive technologies play a major role in the cattle industry. An increase in the use of in vitro-derived embryos is currently being seen around the globe. But the efficiency and quality of the in vitro-derived embryos are substandard when compared to the in vivo production. Different protocols have been designed to overcome this issue, one of those being the use of reproductive fluids as supplementation to embryo culture media. In this study, in vitro-derived calves produced with reproductive fluids added to their embryo production protocol were followed for the first year of life pairwise with their in vivo control, produced by artificial insemination (AI), and their in vitro control, produced with standard supplementation in embryo production. The objective was to assess if any differences could be found in terms of growth and development as well as hematological and biochemical analytes between the different systems. All the analysed variables (physical, hematological, and biochemical) were within physiological range and very similar between calves throughout the entire experiment. However, differences were more evident between calves derived from standard in vitro production and AI. We concluded that the use of reproductive fluids as a supplementation to the embryo culture media results in calves with closer growth and development patterns to those born by AI than the use of bovine serum albumin as supplementation.

Download Full-text