Disappearance and uptake of [125I]FSH in the rat, rabbit, ewe and cow

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D. B. LasterUSDA

Date of this Version

1972

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Published in J. Reprod. Fert. (1972) 30, 407-415

Abstract

Follicle-stimulating hormone (NIH-FSH-S8) was labelled with 125I to determine its disappearance rate after a single intravenous injection and to determine the level of circulating [125I]fsh in the blood after a single intramuscular or subcutaneous injection in the rat, rabbit, ewe and cow. There was a difference in the disappearance and uptake rates among the four species, but the shape of the curve for rate of loss and uptake of labelled fsh was similar in all species. The disappearance of radioactivity occurred at two rates; the first from 1 to 8 min and the second from 16 to 96 min. The half-life, calculated from the total decay curve in each species was 94±21, 118±16, 334±41 and 301±23 min for the rats, rabbits, ewes and cows, respectively. Intramuscular injections resulted in an average of 56% higher [125I]fsh blood levels than subcutaneous injections for all species.

Embryo Transfer in Cattle

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Published on: August 18, 2021

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Reproductive Physiology Review

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In Vitro Fertilization in Cattle

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Articles of Interest

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https://www.animal-reproduction.org/article/5b5a604bf7783717068b46a0

https://www.journalofdairyscience.org/article/S0022-0302(19)30635-6/fulltext

https://www.animal-reproduction.org/article/doi/10.21451/1984-3143-AR1002

https://www.semanticscholar.org/paper/Artificial-Insemination-and-Embryo-Transfer-in-Farin/da20551c1a0fad2bafd1dd931de027691a6bebe0

https://www.sciencedirect.com/science/article/pii/S0022030201746905

https://www.mdpi.com/2076-2615/11/6/1666/htm

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830735/

https://www.sciencedirect.com/science/article/pii/S0022030208712153

https://rep.bioscientifica.com/view/journals/rep/154/6/REP-17-0357.xml

Equine chorionic gonadotropin increases estradiol levels in the bovine oviduct and drives the transcription of genes related to fertilization in superstimulated cows

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Published on: February 18, 2021

Patricia K Fontes 1Eduardo M Razza 1Antônio G R Pupulim 2Ciro M Barros 1Anthony C de Souza Castilho 3

1Departament of Pharmacology, Institute of Biosciences, Universidade Estadual Paulista (UNESP), Botucatu, São Paulo, Brazil.

2Centro Universitário Cesumar (UNICESUMAR), Maringá, Paraná, Brazil.

3Universidade do Oeste Paulista (UNOESTE), Presidente Prudente, São Paulo, Brazil.

https://pubmed.ncbi.nlm.nih.gov/31353672/

Mol Reprod Dev. 2019 Nov;86(11):1582-1591. doi: 10.1002/mrd.23243. Epub 2019 Jul 29.

Abstract

In the bovine oviduct, estradiol (E2) stimulates secretion and cell proliferation, whereas progesterone (P4) suppresses them. In this study, we have evaluated the effect of two superstimulatory protocols (follicle-stimulating hormone [FSH] or FSH combined with equine chorionic gonadotropin [eCG]) on the oviductal levels of E2 and P4 and its outcome on oviductal cells. Compared with the control group (a single pre-ovulatory follicle), we have observed that the cows submitted to FSH/eCG treatment showed a higher concentration of E2 in the oviduct tissue, together with a higher abundance of messenger RNA encoding steroid receptors (ESR1 and progesterone receptor), and genes linked to gamete interactions and regulation of polyspermy (oviduct-specific glycoprotein 1, heat-shock protein family A member 5, α-l-fucosidase 1 [FUCA1], and FUCA2) in the infundibulum and ampulla segments of the oviduct. However, we did not observe any modulation of gene expression in the isthmus segment. Even though the FSH protocol upregulated some of the genes analyzed, we may infer that the steady effect of FSH combined with eCG on oviduct regulation might benefit fertilization and may potentially increase pregnancy rates.

Keywords: cattle; female reproductive tract; gametes; gene expression; steroids; superovulation.

Effect of superstimulation on the expression of microRNAs and genes involved in steroidogenesis and ovulation in Nelore cows

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Published on: February 18, 2021

P H Santos 1R A Satrapa 2P K Fontes 1F F Franchi 1E M Razza 1F Mani 3M F G Nogueira 4C M Barros 1A C S Castilho 5

1Universidade Estadual Paulista (UNESP), Institute of Biosciences, Campus Botucatu, Department of Pharmacology, Botucatu, São Paulo, Brazil.

2Universidade Federal do Acre (UFAC), Center of Biological and Natural Sciences, Rio Branco, Acre, Brazil.

3Universidade Estadual Paulista (UNESP), Institute of Biosciences, Campus Botucatu, Department of Chemistry and Biochemistry, Botucatu, São Paulo, Brazil.

4Universidade Estadual Paulista (UNESP), School of Sciences and Languages, Campus Assis, Department of Biological Sciences, Assis, São Paulo, Brazil.

5Universidade Estadual Paulista (UNESP), School of Sciences and Languages, Campus Assis, Department of Biological Sciences, Assis, São Paulo, Brazil. Electronic address: anthony@unoeste.br.

https://pubmed.ncbi.nlm.nih.gov/29407901/

Theriogenology 2018 Apr 1;110:192-200. doi: 10.1016/j.theriogenology.2017.12.045. Epub 2018 Jan 11.

Abstract

To better understand the impact of ovarian superstimulation on bovine follicular microenvironment, Nelore cows (Bos taurus indicus) were subjected to ovarian superstimulation with follicle stimulating hormone (FSH, n = 10; P-36 protocol) or FSH combined with eCG (n = 10; P-36/eCG protocol). Follicular fluid was analyzed for cholesterol concentration. Granulosa cells were analyzed by RT-qPCR to assess the expression of genes involved in steroidogenic and ovulatory and expression of microRNAs involved in final follicular development and luteinizing hormone/choriogonadotropin receptor (LHCGR) expression. Plasma concentration of estradiol was also measured. Follicular fluid from the P-36 group showed higher concentration of cholesterol than that of control (non-superstimulated) cows. Plasma concentration of estradiol was higher in the P-36/eCG group. Abundance of STAR and FSHR mRNAs were lower in granulosa cells from the P-36/eCG group. In contrast, LHCGR mRNA abundance was higher in superstimulated granulosa cells from the P-36 group and showed a pattern opposite to that of miR-222 expression. Ovarian superstimulation did not affect the expression of other markers (mmu-miR-202-5p, has-miR-873, has-miR-144, and their target genes, CREB, TGFBR2, and ATG7) of antral follicle development. However, the mRNA expression of VEGF pathway components was modulated by P-36 treatment. Taken together, these results demonstrate that superstimulatory protocols modify steroidogenic capacity, increase plasma estradiol, and regulate the abundance of VEGF system, LHCGR mRNA and suppress the expression of miR-222 in bovine granulosa cells.

The incompletely fulfilled promise of embryo transfer in cattle—why aren’t pregnancy rates greater and what can we do about it?

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Published on: February 18, 2021

Peter J Hansen

Department of Animal Sciences, D.H. Barron Reproductive and Perinatal Biology Research Program, and Genetics Institute, University of Florida, Gainesville, FL

https://academic.oup.com/jas/article/98/11/skaa288/5954183

Journal of Animal Science, Volume 98, Issue 11, November 2020, skaa288 https://doi.org/10.1093/jas/skaa288

Abstract

Typically, bovine embryos are transferred into recipient females about day 7 after estrus or anticipated ovulation, when the embryo has reached the blastocyst stage of development. All the biological and technical causes for failure of a female to produce a blastocyst 7 d after natural or artificial insemination (AI) are avoided when a blastocyst-stage embryo is transferred into the female. It is reasonable to expect, therefore, that pregnancy success would be higher for embryo transfer (ET) recipients than for inseminated females. This expectation is not usually met unless the recipient is exposed to heat stress or is classified as a repeat-breeder female. Rather, pregnancy success is generally similar for ET and AI. The implication is that either one or more of the technical aspects of ET have not yet been optimized or that underlying female fertility that causes an embryo to die before day 7 also causes it to die later in pregnancy. Improvements in pregnancy success after ET will depend upon making a better embryo, improving uterine receptivity, and forging new tools for production and transfer of embryos. Key to accelerating progress in improving pregnancy rates will be the identification of phenotypes or phenomes that allow the prediction of embryo competence for survival and maternal capacity to support embryonic development.

‘There is only one thing that is truly important in an IVF laboratory: everything’ Cairo Consensus Guidelines on IVF Culture Conditions

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Published on: February 18, 2021

Cairo Consensus Group

https://www.sciencedirect.com/science/article/pii/S1472648319307540#!

Reproductive BioMedicine Online Volume 40, Issue 1, January 2020, Pages 33-60

Abstract

This proceedings report presents the outcomes from an international expert meeting to establish consensus guidelines on IVF culture conditions. Topics reviewed and discussed were: embryo culture – basic principles and interactions; temperature in the IVF laboratory; humidity in culture; carbon dioxide control and medium pH; oxygen tension for embryo culture; workstations – design and engineering; incubators – maintaining the culture environment; micromanipulation – maintaining a steady physcochemical environment; handling practices; assessment practices; culture media – buffering and pH, general composition and protein supplementation, sequential or single-step media for human embryo culture; use and management – cold chain and storage; test equipment – calibration and certification; and laboratory equipment and real-time monitoring. More than 50 consensus guideline points were established under these general headings.

Keywords: Culture conditions, Incubators, IVFMaintenance, Medium, Quality control

Direct transfer of frozen-thawed bovine embryos and its application in cattle reproduction management

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Published on: February 18, 2021

Osamu DOCHI1

1)Rakuno Gakuen University, Hokkaido 069-0851, Japan

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6815740/

J Reprod Dev. 2019 Oct; 65(5): 389–396.Published online 2019 Jun 13. doi: 10.1262/jrd.2019-025

Abstract

Embryo transfer entails many procedures and techniques, of which embryo freezing is an important component in bovine embryo transfer. Embryo freezing techniques have been developed over the last 40 years, allowing practical availability, and have become essential for cattle reproduction management under field conditions. The direct transfer methods of frozen-thawed, in vivo-derived, and in vitro-produced (IVF) bovine embryos using 1.5 M ethylene glycol (EG) with or without sucrose (SUC) are used widely under on-farm conditions, not only in Japan but also globally. The direct transfer method using 1.5 M glycerol (GLY) and 0.25 M SUC (GLY-SUC) is used mainly in Japan. The pregnancy rate with direct transfer of frozen-thawed bovine embryos in either EG or GLY-SUC has been found to not differ from conventional freezing with GLY and traditional dilution techniques. Pregnancy rates following direct transfer of frozen-thawed bovine embryos were affected by the developmental stage of the embryos and the parity of the recipients. The use of ultrasound-guided on-farm ovum pickup is ushering in a new revolution for the commercial application of IVF embryos. Globally, for the first time more IVF bovine embryos were transferred in 2017 than produced in vivo. More than 60% of IVF embryos were transferred fresh due to a low pregnancy rate of frozen-thawed IVF embryos. Many factors seemed to be involved in improving the survival rate of frozen-thawed IVF embryos. Therefore, further research is needed to improve the freezing tolerance of IVF embryos to develop efficient direct transfer methods analogous to those used for in vivo embryos.

Keywords: Bovine embryo, Direct transfer, Ethylene glycol, Freezing, Pregnancy rate

Articles of Interest

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Published on: February 18, 2021

Ano-genital distance relates to fertility

Effect of dose and timing of prostaglandin F2α treatments during a 7-d Ovsynch protocol on progesterone concentration at the end of the protocol and pregnancy outcomes in lactating Holstein cows

Estradiol cypionate administered at the end of a progesterone-based protocol for FTAI induces ovulation and improves postovulatory luteal function and uterine environment in anestrous beef cows

Association between heat stress during intrauterine development and the calving-to-conception and calving-to-first-service intervals in Holstein cows

Impact of ethanol and heat stress–dependent effect of ultra-diluted Arnica montana 6 cH on in vitro embryo production in cattle

Analysis of bovine blastocysts indicates ovarian stimulation does not induce chromosome errors, nor discordance between inner-cell mass and trophectoderm lineages

Developmental kinetics and viability of bovine embryos produced in vitro with sex-sorted semen

Heat stress and reproduction – A foreword

Growing cattle embryos beyond Day 8 – An investigation of media components

Melatonin enhances in vitro developmental competence of cumulus-oocyte complexes collected by ovum pick-up in prepubertal and adult dairy cattle

Effect of expression of estrus and treatment with GnRH on pregnancies per AI in beef cattle synchronized with an estradiol/progesterone-based protocol

The incompletely fulfilled promise of embryo transfer in cattle—why aren’t pregnancy rates greater and what can we do about it?

2021 IETS proceedings

Effects of epidermal growth factor and progesterone on oocyte meiotic resumption and the expression of maturation-related transcripts during prematuration of oocytes from small and medium-sized bovine antral follicles              

Effect of Supplemental Trace Minerals on Standard and Novel Measures of Bull Fertility

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Published on: November 2, 2020

T. W. Geary – a2, R. C. Waterman – a, M. L. Van Emon – b, C. R. Ratzburg – c, S. Lake – c, B. A. Eik – a, D. R. Armstrong – a, A. L. Zezeski – a, and J. S. Heldt – d

aUSDA-ARS, Fort Keogh Livestock and Range Research Laboratory, Miles City, MT 59301; bDepartment of Animal and Range Sciences, Montana State University, Bozeman, MT 59717; cDepartment of Animal Sciences, University of Wyoming, Laramie, WY; dMicronutrients USA LLC, 2601 Fortune Circle Drive E. Suite 200C, Indianapolis, IN 46241

Abstract

Two studies were conducted to evaluate the effects of trace mineral supplementation on traditional and novel measures of bull fertility. In experiment 1, 37 mature bulls received one of three dietary supplements daily for 71 d: 1) Supplement without Cu, Zn, and Mn (CON); 2) Supplement with Cu, Zn, and Mn sulfate (SULF); and 3) Supplement with basic Cu chloride, and Zn and Mn hydroxychloride (CHLR). In experiment 2, 128 Angus or Angus-Hereford calves were maintained on a growing diet for 75 d (year 1) or 119 d (year 2) in Calan gate equipped pens without mineral supplementation. Bulls (n = 32 head/treatment) received one of four trace mineral supplements daily for 84 d: 1) Zn with no Cu (ZN), 2) Cu with no Zn (CU), 3) Cu and Zn (ZNCU), or 4) no Cu or Zn (CON). Fertility measures included a breeding soundness examination (BSE) and novel fertility measures conducted using flow cytometry. In mature bulls, final liver Zn concentration was positively correlated (P = 0.02) with sperm concentration (r = 0.31) and tended (P = 0.06) to be negatively correlated with acrosome damage (r = -0.39). Peripubertal bulls receiving ZNCU had greater ADG than CU bulls (P = 0.05). Each BSE and novel fertility component improved from d 0 to 84 in peripubertal bulls and were not affected (P > 0.10) by mineral supplementation. Bulls that received no supplement (CON) had greater (P < 0.01) percentage of sperm with distal midpiece reflex and Dag defect in their ejaculates. Sperm viability after 30 min of incubation were not affected by trace mineral supplementation, but after 3 h incubation, sperm viability tended to differ (P = 0.06) between treatments and tended to be less for CON bulls compared to ZNCU bulls. Among contrast comparisons, trace mineral supplemented bulls had greater (P < 0.05) percentage of viable sperm at 3 h post collection and reactive oxygen resistant sperm than CON bulls. Addition of Zn to trace mineral containing Cu (ZNCU) improved (P < 0.05) percentage of sperm in the ejaculate with high mitochondrial energy potential and viable sperm with intact acrosome membrane. In summary, it appears the homeostasis mechanisms for bull trace mineral maintenance are extremely efficient and mineral supplementation of mature and peripubertal bulls did not have major improvements in any laboratory or chute-side measures of bull fertility, however bulls exposed to breeding or in environments with diet antagonists might respond differently.

Out of Season Artificial Insemination and Embryo Transfer Results in Ewes: A Field Trial

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Published on: November 2, 2020

B Price, T Mittleider, S Collins, P Gibbons, and J Gibbons

College of Veterinary Medicine, Lincoln Memorial University, Harrogate, TN

Introduction: 

Ewes are seasonally polyestrous short-day breeders with an estrous cycle of approximately 16 – 17 days.  In the northern hemisphere ewes have active estrous cycles and are naturally receptive to rams from late September to late December when there is less than 12 hours of daylength.  However, progressive sheep breeders often prefer to breed sheep earlier in the year, during periods where there is more than twelve hours of daylength, (July and August) in order to have lambs that are appropriate to target specific show markets.  In order to facilitate this out of season breeding and accelerate genetic gain, producers rely on Assisted Reproductive Techniques such as Laparoscopic Artificial Insemination (LAI), ovarian hyper-stimulation, embryo collection from valuable embryo donors, and embryo transfer (ET) into synchronized recipients (1,2,3.)  This field trial was conducted during late July through early August in southwest Virginia (latitude 36-38’12” N), during a daylight period of about 14 hours. Pregnancy rates of ewes bred by means of AI were compared to those that underwent ET.

Hatching Blastocysts

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Published on: November 2, 2020

Day 7 in vitro produced bovine hatching blastocysts.

By Dr Daniela Demetrio – Ruann Genetics – Riverdale, CA

7 & 7 Synch: An Estrus Synchronization Protocol for Postpartum Beef Cows

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Published on: November 2, 2020

Overview


Researchers at the University of Missouri recently
evaluated a new protocol for synchronization of estrus
among postpartum beef cows. This protocol was found
to be highly effective both for cows receiving embryo
transfer (ET) and cows receiving fixed-time artificial
insemination (AI). Extensive field trials with 7 & 7
Synch observed improvements in the proportion of
cows expressing estrus and in the proportion of cows
becoming pregnant to embryo transfer or to AI.

How can we improve embryo production and pregnancy outcomes of Holstein embryos produced in vitro? (12 years of practical results at a California dairy farm)

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Published on: November 2, 2020

Daniela Garcia Borges Demetrio, 1 ,* Eduardo Benedetti, 2 Clarice Garcia Borges Demetrio, 3 Julio Fonseca, 1 Mayara Oliveira, 1 Alvaro Magalhaes, 1 and Ricarda Maria dos Santos 4

1RuAnn Genetics, Riverdale, CA, United States
2Arizona Dairy Co, Mesa, AZ, United States
3Escola Superior de Agricultura “Luiz de Queiroz”, Universidade do Estado de São Paulo, Piracicaba, SP, Brasil
4Faculdade de Medicina Veterinária, Universidade Federal de Uberlândia, Uberlândia, MG, Brasil

Abstract


Genomic evaluations have revolutionized dairy cattle breeding, and the demand for embryos produced from very young heifers with high genetic merit has increased over time. The combination of low oocyte recovery, young age of donors, and milk production status can make the in vitro embryo production (IVP) of Holstein cattle incredibly challenging. Several factors need to be coordinated to obtain a live calf from an IVP embryo, but the quality of the oocyte at the start of the process is one of the key factors. Aspects related to oocyte quality, laboratory quality control, embryo quality and recipient selection are addressed here, based on the measures that the RuAnn Genetics Laboratory (Riverdale, California, USA) adopted in the last 12 years, with the goal of improving production of live, healthy calves from Holstein embryos. Follicular wave synchronization and stimulation with follicular stimulating hormone (FSH) is necessary to improve oocyte quality and consequently embryo production. Laboratory quality control and the use of high-quality supplies are essential to reduce variability in production and facilitate identification of other factors that might interfere with embryo production. High pregnancy rates can be achieved with good quality embryos selected at optimal time and stage of development, transferred by an experienced embryo transfer (ET) technician, to well managed recipients 7 or 8 days after estrus. Attention to detail at every step of the process is crucial to success.
Keywords: embryo, Holstein, in vitro production, pregnancy, recipient.

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One-step automated bioprinting-based method for cumulus-oocyte complex microencapsulation for 3D in vitro maturation

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Published on: November 2, 2020

Antonella Mastrorocco1¤a, Ludovica Cacopardo2, Nicola Antonio Martino1¤b, Diana Fanelli3, Francesco Camillo3, Elena Ciani1, Bernard A. J. Roelen4, Arti Ahluwalia2,5☯, Maria Elena Dell’Aquila1☯

1 Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari Aldo Moro, Bari, Italy; 2 Research Centre E. Piaggio, University of Pisa, Pisa, Italy; 3 Department of Veterinary Sciences, University of Pisa, Pisa, Italy; 4 Department of Clinical Sciences, Embryology, Anatomy and Physiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands; 5 Department of Information Engineering, University of Pisa, Pisa, Italy

☯ These authors contributed equally to this work.
¤a Current address: Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy
¤b Current address: Department of Veterinary Sciences, University of Torino, Torino, Italy

Abstract

Three-dimensional in vitro maturation (3D IVM) is a promising approach to improve IVM efficiency as it could prevent cumulus-oocyte complex (COC) flattening and preserve its structural and functional integrity. Methods reported to date have low reproducibility and validation studies are limited. In this study, a bioprinting based production process for generating microbeads containing a COC (COC-microbeads) was optimized and its validity tested in a large animal model (sheep). Alginate microbeads were produced and characterized for size, shape and stability under culture conditions. COC encapsulation had high efficiency and reproducibility and cumulus integrity was preserved. COC-microbeads underwent IVM, with COCs cultured in standard 2D IVM as controls. After IVM, oocytes were analyzed for nuclear chromatin configuration, bioenergetic/oxidative status and transcriptional activity of genes biomarker of mitochondrial activity (TFAMATP6ATP8) and oocyte developmental competence (KHDC3NLRP5OOEP and TLE6). The 3D system supported oocyte nuclear maturation more efficiently than the 2D control (P<0.05). Ooplasmic mitochondrial activity and reactive oxygen species (ROS) generation ability were increased (P<0.05). Up-regulation of TFAMATP6 and ATP8 and down-regulation of KHDC3NLRP5 expression were observed in 3D IVM. In conclusion, the new bioprinting method for producing COC-microbeads has high reproducibility and efficiency. Moreover, 3D IVM improves oocyte nuclear maturation and relevant parameters of oocyte cytoplasmic maturation and could be used for clinical and toxicological applications.

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Sperm DNA Integrity and Male Fertility in Farm Animals: A Review

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Published on: November 2, 2020

Arumugam Kumaresan1*, Mohua Das Gupta1, Tirtha Kumar Datta2 and Jane M. Morrell3

  • 1Theriogenology Laboratory, Southern Regional Station of National Dairy Research Institute (ICAR), Bengaluru, India
  • 2Animal Genomics Laboratory, National Dairy Research Institute (ICAR), Karnal, India
  • 3Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden

The accurate prediction of male fertility is of major economic importance in the animal breeding industry. However, the results of conventional semen analysis do not always correlate with field fertility outcomes. There is evidence to indicate that mammalian fertilization and subsequent embryo development depend, in part, on the inherent integrity of the sperm DNA. Understanding the complex packaging of mammalian sperm chromatin and assessment of DNA integrity could potentially provide a benchmark in clinical infertility. In the era of assisted reproduction, especially when in-vitro fertilization or gamete intrafallopian transfer or intracytoplasmic sperm injection is used, assessment of sperm DNA integrity is important because spermatozoa are not subjected to the selection process occurring naturally in the female reproductive tract. Although sperm DNA integrity testing measures a significant biological parameter, its precise role in the infertility evaluation in farm animals remains unclear. In this review, the earlier findings on sperm DNA integrity in relation to male fertility are compiled and analyzed. Furthermore, the causes and consequences of sperm DNA damage are described, together with a review of advances in methods for detection of sperm DNA damage, and the prognostic value of sperm DNA quality on male fertility.

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Short term incubation of frozen/thawed bovine embryos

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Published on: August 13, 2020

Dalena Hobbs, Colton Holcomb, and John Gibbons, College of Veterinary Medicine, Lincoln Memorial University, Harrogate, TN, 37752

Introduction:

Embryo transfer is an assisted reproductive technique that enables progressive cattle producers to reach their financial, reproductive, and genetic goals and the process began to gain considerable traction in the late 1970s to early 1980s as non-surgical methods to collect embryos were developed (Troxel, 2013).  Currently, conventional embryo collection techniques require ovarian hyper-stimulation of donor cows with exogenous FSH, artificial insemination, and trans-cervical uterine lavage to recover embryos about 7 days post insemination.  Another approach that has gained popularity is in vitro fertilization (Mapletoft, 2013).  In vitro embryo production involves recovering the ova directly from the ovaries using an ultrasound guided trans-vaginal follicular aspiration technique.  The recovered oocytes are matured, fertilized, and cultured in vitro and this approach has become substantially popular recently despite the increased cost associated with specialized equipment, training, staff, etc.  According to AETA reports (AETA, 2017; 2019) there has been a 120% increase in the number of in vitro produced embryos transferred over the most recent two years of available AETA data (AETA, 2017; 2019).  In vitro embryo production seems to be gaining popularity over conventional embryo transfer techniques because of the potential to produce more calves per year (Stadheim, 2015), may require fewer hormone injections, and does not require synchronization.  In conventional in vivo embryo collections, almost one half of recovered ova are non-viable, and that percentage has not changed substantially in many years (AETA, 2010; 2019).  Many of these non-viable ova are considered degenerate embryos that have not developed to the appropriate stage relative to the other embryos in the cohort. Salvaging these degenerate embryos that would otherwise be discarded may translate to additional embryo transfers or calves per embryo collection.  This experiment evaluated short term incubation environments and the potential damage to the embryo and zona pellucida associated with the freeze / thaw process.  As many practitioners have become involved in in vitro embryo production, the equipment, supplies, and staff are likely in place to consider an in vitro culture approach of degenerate or poor quality embryos (fresh or post-thaw) to enable the development of these embryos.  

Methods and Materials:

Frozen / thawed bovine (Day 7) in vivo derived embryos processed for direct transfer (Ethylene Glycol) were thawed for 30 seconds in a 30°C water bath. Thawed embryos (total of 30 / group over three replicates) were placed in commercially available holding media temporarily to be graded and staged (according to the International Embryo Technology Society rubric) and then placed into either holding media, phosphate buffered saline (PBS) supplemented with 15% fetal bovine serum (FBS; v/v) and antibiotic / antimycotic (gentamicin; 2 mL/ml; v/v), or a commercially available in vitro culture media for approximately 18 hours at 38.5°C. Embryos in the holding media and PBS (+15% FBS) were loaded (individually) into ¼ cc plastic straws which were sealed and submerged in a water bath (38.5°C). Embryos in the in vitro culture media group were rinsed and placed (individually) in equilibrated 25 mL culture drops on tissue coated plastic 60 mm dishes overlaid with lightweight mineral oil and were incubated in 5% CO2 and 100% humidified air (18 hours). Following the incubation period, embryos were recovered, rinsed, and graded and staged again. The numerical change in the embryo grade (1 through 4) and stage (3 through 8) from the pre-freeze information on the straw label, the post-thaw and post-incubation evaluation were recorded and analyzed with ANOVA.

Results:

Statistically, there was no difference between the pre-freeze, post-thaw, and post-incubation grades or stages between the holding media and PBS+FBS group; however, there was a decrease in the quality grade (P<0.001) following incubation in all groups (Figure 1).  The decline in the quality grade following incubation in the holding media and PBS+FBS groups was significantly lower than the decline in the quality grade in the in vitro culture group (Figure 1).  There was also a significant decline in the quality grade associated with the freeze / thaw cycle among all the groups (Figure 1).  The developmental stage pre-freeze and post-thaw and post incubation was unaffected in the holding media and PBS+FBS groups; however, in the in vitro culture group, on average embryos developed from the morula to the early blastocyst (P<0.001) stage indicating that on average, viability was maintained. This experiment also indicated that approximately 29% of embryos experience some form of damage to the zona pellucida following the freeze / thaw process.

Conclusion:

Cryopreservation of bovine embryos – although critically important to the embryo transfer industry (Mapletoft, 2013), is detrimental to the quality of the embryo and zona pellucida.  With the advent of the direct transfer technology, this decrease in quality is not obvious as embryos are seldom observed post thaw.  Practically, incubation of poor quality embryos for some time may be a mechanism to salvage a few embryos that have not reached the developmental stage of other embryos in the collection and are normally discarded.  The in vitro culture media and system provided a substantially more effective environment to enable embryos to develop further, although the quality of those embryos was negatively affected.  It is difficult to determine if the damaging effects of the freeze / thaw cycle can be overcome during an incubation period; however, the damage was apparently suppressed using an in vitro culture approach.  Holding media and PBS+FBS while useful as a temporary storage device for bovine embryos is not an adequate short-term incubation media and apparently did not mitigate any damage due to the freeze / thaw process.  Future research will involve short-term incubation of fresh embryos in order to eliminate the negative effects associated with cryopreservation.  In conclusion, these results and future research may be useful in the bovine embryo industry, and for cattle producers alike, by increasing the number of transferable quality embryos that would otherwise be discarded.

References:

AETA Statistics Committee Report – 2010

AETA Statistics Committee Report – 2017

AETA Statistics Committee Report – 2019

Mapletoft, R. (2013, September). History and Perspectives on Bovine Embryo Transfer. Retrieved June 22, 2020, from https://www.animal-reproduction.org/article/5b5a6048f7783717068b468e/pdf/animreprod-10-3-168.pdf

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Ovarian profile and pregnancy rates following ovulation synchronization and timed-artificial insemination in dairy cows

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Published on: August 13, 2020

a Megan Bollman, b Ashley Greenhawk, b Ann Shipley, a Philippa Gibbons, a John Gibbons

a College of Veterinary Medicine, Lincoln Memorial University, Harrogate, TN 37752, b Hickory Corner Dairy, Speedwell, TN 37870

Introduction:

In the dairy reproduction industry, determining the precise timing for artificial insemination (AI) is a crucial component in obtaining a successful pregnancy outcome. The detection of estrus in dairy cattle is typically characterized by visible behavioral signs such as increased activity and vocalization, aggressive behavior, mounting, and standing to be mounted (Reith & Hoy, 2018).  Recognition of estrus has historically been difficult due to behavioral variability among individual animals and environment. The appearance and duration of estrus can be influenced by high milk yield, inadequate nutrition, stress, and overall welfare of an individual animal (Nowicki, Baraniski, Baryczka, & Janowski, 2017).  It is also time consuming and expensive for farm staff to monitor the herd for these behavioral signs. Recognition of estrus still remains low even though reproduction management technology strategies, i.e. pressure sensing systems, video cameras, activity meters; have been implemented to ease the task of visually identifying estrus in dairy cattle, (Reith, & Hoy, 2018). Previous studies have shown that a range of 50% of cattle in estrus exhibiting behavioral signs were identified with visual observation to 70% of cattle in estrus were identified using an activity monitoring system (Carvalho et al., 2014). Without the proper technology or technique for estrus detection, strategies to adequately time artificial insemination continue to be a challenge to the dairy industry.

Newer technologies such as timed artificial insemination has been widely used following the synchronization of ovulation in dairy cattle (Wiltbank, & Pursley, 2014). Ovulation synchronization eliminates the need to recognize estrus prior to artificial insemination. Since its introduction in 1995 Ovsynch® and its newer modifications, Presynch-Ovsynch® and Double-Ovsynch®, have almost replaced estrus detection in many dairy herds (Carvalho et al., 2014). By manipulating hormones in order to synchronize ovulation, the challenges of visual estrus identification are reduced, and the number of dairy cattle serviced through timed artificial insemination is increased (Nowicki, Baraniski, Baryczka, & Janowski, 2017). Ovsynch® and its modified protocols may be useful to improve reproduction performance in dairy cattle as it facilitates by appointment breeding and some dairy cows that showed no signs of estrus will indeed be serviced and become pregnant.

The focus of this case study was to evaluate the hormonal response of a dairy herd by observing their ovarian structures following a modified Ovsynch® protocol.  The ovarian structures were observed on the day of insemination and retrospectively correlated to pregnancy outcome.

Methods:

A modified Ovsynch® protocol was implemented at a large (≈ 700 cows) local dairy, and is illustrated in Figure 1.  Data was collected from lactating dairy cattle from November 28th, 2018 through May 24th, 2019.

On the day of AI, transrectal ultrasonography was conducted to observe ovarian structures. Follicular and corpora lutea (CL) structures were visualized measured and data recorded to retrospectively relate ovarian structures with the pregnancy status on Day 35 post AI. Insemination was conducted regardless of ovarian status, by a single technician using commercially available frozen semen. On Day 35 after AI, transrectal ultrasonography was again used to observe the presence of uterine fluid, ovarian structures, abnormal findings, and to detect the presence of a viable fetus

Results and Discussion:

A total of 60 out of 148 lactating dairy cattle that were analyzed successfully became pregnant following a modified Ovsynch® protocol, giving an overall pregnancy rate of 40.5 ± 0.04% (Table 1). However, the diameter of the largest follicle was not significantly different (P>0.05) between those cows that became pregnant (18.0 ± 0.6mm), and those that did not become pregnant (18.1 ± 0.5mm). Putative cystic cows (largest follicle > 30 mm) were excluded from this analysis; however, 3 of the 6 cows considered to be cystic but were inseminated became pregnant (Largest follicle diameter = 35.3 ± 0.9 versus 40.0 ± 5.0 mm, pregnant versus open). The presence of CL structures in cattle that became pregnant and cattle that did not become pregnant was similar (P>0.05; 25.0 and 29.5%, respectively; Table 1). The diameter of the CL was also similar (P>0.05) between those cows that became pregnant, and those that did not (17.3 ± 1.3 and 16.7 ± 1.0mm, respectively; Table 1).

The average diameters of the ovarian structures (Follicles =18mm, CL =17mm) in lactating dairy cows that became pregnant verse those that did not were further investigated.  In a higher (P=0.056) percentage of pregnant cows, the diameter of the largest follicle was ≤18mm (65.0 ± 0.1%) compared to those cows in which the largest follicle was >18mm (35.0 ± 0.1%; Table 2).  Although numerically superior, there was no statistical difference in the percentage of pregnant cows with a CL diameter of <17mm (60.0 ± 0.2%) compared to those with a CL >17mm (40.0 ± 0.1%; Table 2).

There was a trend (P=0.131) for a higher percentage of the non-pregnant cows to have a diameter of the largest follicle ≤18mm (55.7 ± 0.1%) compared to those in which the largest follicle >18mm (44.3 ± 0.1%; Table 2).  There was no statistical difference in the percentage of non-pregnant cows with a CL diameter of <17mm (52.0 ± 0.1%) compared to those with a CL >17mm (48.0 ± 0.1%; Table 2).

Conclusion:

The use of timed artificial insemination programs and transrectal ultrasonography are beneficial in reproduction management strategies (Colazo, & Mapletoft, 2014). Ovulation synchronization in lactating dairy cows has continued to be an efficient management tool in the dairy reproduction industry. The analysis of ovarian structures following a modified Ovsynch® protocol was useful but not absolute in predicting which cows would become pregnant and which would not.  This study determined that although the diameter of the largest follicle in dairy cows at AI did not influence pregnancy rate, a higher percentage of cows that became pregnant had smaller follicles (≤18mm).  Any effects of the presence or diameter of the CL on pregnancy status was apparently outweighed by other factors.  Further, it is unclear whether the cows that did not become pregnant failed to respond to the synchronization process or were influenced by these factors (nutrition, stress, lactational status, body condition, genetics, etc.).  In addition to evaluating overall reproductive health, trans-rectal ultrasonography may be a useful tool for predicting pregnancy outcome.  Further research is required to evaluate a more robust ovarian classification system or to evaluate of the endocrine status at the time of AI may also be useful to determine which dairy cows will likely become pregnant or not following Ovsynch® and AI.

Acknowledgements:

The Authors appreciate the assistance and access to the lactating dairy cows provided by Hickory Corner Dairy, Speedwell, TN

References:

Carvalho, P.D., Guenther, J.N., Fuenzalida, M.J., Amundson, M.C., Wiltbank, M.C., Fricke, P.M. (2014). Presynchronization using a modified Ovsynch protocol or a single gonadotropin-releasing hormone injection 7 d before an Ovsynch-56 protocol for submission of lactating dairy cows to first timed artificial insemination. Journal of Dairy Science. 97(10), 6305-6315. Retrieved from: https://www.sciencedirect.com/science/article/pii/S0022030214005244#bib0210

Colazo, Marcos G., & Mapletoft, Teuben J. (2014). A review of current timed-AI (TAI) programs for beet and dairy cattle. The Canadian Veterinary Journal. 55(8), 772-780. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4095965/

Nowicki, A., Baraniski, W., Baryczka, A., & Janowski, T. (2017). Ovsynch protocol and its modifications in the reproduction management of dairy cattle herds-an update. Journal of Veterinary Research. 61(3), 329-336. Retrieved from https://content.sciendo.com/view/journals/jvetres/61/3/article-p329.xml

Reith, S., & Hoy, S. (2018). Review: Behavioral signs of Estrus and the Potential of Fully Automated systems for Detection of Estrus in Dairy Cattle. NCBI 12(2), 398-407. Retrieved from https://pubmed.ncbi.nlm.nih.gov/28807076/

Wiltbank, Milo C., & Pursley, Richard J. (2014). The cow as an induced ovulatory: Timed AI after synchronization of ovulation. Theriogenology 81(1), 170-185. Retrieved from https://www.sciencedirect.com/science/article/pii/S0093691X13003828

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