Curtis R. Youngs

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 – 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6°C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5°C/min, enabling further dehydration, to a temperature of -34°C before being plunged into liquid nitrogen (-196°C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37°C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.

Assisted Reproductive Technologies and Embryo Culture Methods for Farm Animals

Assisted reproductive technologies have played a major role in the genetic improvement of livestock as well as in the development of transgenic animal technologies. Artificial insemination (AI) was the first widely-accepted assisted reproductive technology for livestock, and this technology allows genetically superior males to produce more offspring than would be possible through conventional mating. Conventional AI has been enhanced in recent years with the use of sperm cells that have been flow cytometrically sorted into populations of X chromosome- and Y chromosome-bearing spermatozoa, facilitating production of offspring of a pre-determined genetic sex. A related genetic improvement technology, embryo transfer, involves non-surgical recovery of preimplantation embryos from one female and subsequent transfer into recipient females to enable genetically superior females to produce a greater number of offspring than would be possible through traditional mating. With the advent of ultrasound-guided procedures for recovery of oocytes from living females, coupled with advances in in vitro oocyte maturation, in vitro fertilization and in vitro embryo culture, the production of embryos in vitro is rapidly growing and may soon generate more embryos than are derived in vivo. Embryo micromanipulation procedures such as embryo bisection, microfertilization, and assisted embryo hatching are providing additional tools for reproductive biologists to produce live offspring. Embryo biopsy is permitting preimplantation genetic diagnosis not only for genetic sex determination but also for identification of embryonic genetic defects. Embryo culture and embryo coculture systems have also enabled proliferation of novel assisted reproductive technologies such as oocyte reconstruction and nuclear transfer.

Seroprevalence and risk factors for Coxiella burnetii , the causative agent of Q fever in the dromedary camel ( Camelus dromedarius ) population in Algeria

Query (Q) fever is a globally distributed zoonotic disease caused by Coxiella burnetii, a bacterial agent for which ruminants are the most prevalent natural reservoir. Data regarding Q fever infection in camels in Algeria are limited. Therefore, a survey to detect seroprevalence of C. burnetii antibodies was conducted among healthy camel populations in a vast area in southeastern Algeria to determine distribution of the Q fever causative organism and to identify risk factors associated with infection. Between January and March 2016, blood samples were collected from 184 camels and serum samples were subsequently analysed using a commercial Enzyme-Linked Immunosorbent Assay (ELISA) kit. At the time of blood collection, a questionnaire investigating 13 potential predisposing factors associated with C. burnetii seropositivity was completed for every dromedary camel and herd. Results were analysed by a chi-square (χ2) test and multivariate logistic regression. The seroprevalence of C. burnetii at the animal level was 71.2% (95% CI: 65.2–78.3) and 85.3% (95% CI: 72.8–97.8) at the herd level. At the animal level, differences in seroprevalence were observed because of herd size, animal age, animal sex, presence of ticks and contact with other herds. A multivariable logistic regression model identified three main risk factors associated with individual seropositivity: (1) age class > 11 years (OR = 8.81, 95% CI: 2.55–30.41), (2) herd size > 50 head (OR = 4.46, 95% CI: 1.01–19.59) and (3) infestation with ticks (OR 2.2; 95% CI: 1.1–4.5). This study of seroprevalence of C. burnetii infection in camels in Algeria revealed a high seroprevalence of Q fever in camel populations in southeastern Algeria and provided strong evidence that Q fever represents an economic, public health and veterinary concern. Appropriate measures should be taken to prevent the spread of C. burnetii and to reduce the risk of Q fever in farm animals and humans in this agro-ecologically and strategically important region of North Africa.

Embryo Transfer Technologies in Pigs

Embryo transfer (ET) became a reality in the swine industry with the birth of the first live ET piglets in 1950. Since that pioneering achievement more than 68 years ago, significant developments in porcine ET and its related technologies have occurred. Although the volume of commercial ET activity with pigs is low compared to that reported for cattle, substantial porcine ET activity is taking place in private companies and institutes engaged in biomedical research. In vitro production of pig embryos has greatly surpassed that of in vivo-derived embryos, and development of nonsurgical methods for transfer of swine embryos has opened the door to potential widespread commercial application of porcine ET. The historical inability to cryopreserve pig embryos has been overcome to a great extent with development of protocols for vitrification of porcine embryos. The creation of genetically modified pigs via somatic cell nuclear transfer or genome-editing technologies depends upon successful ET, and the needs of the biomedical research community likely will be the impetus for further refinements in pig ET technologies.

99 BIRTH OF THE FIRST BOVINE EMBRYO TRANSFER CALF IN THE REPUBLIC OF KOSOVA

The objective of this applied field study was to assess the feasibility of successfully performing bovine embryo transfer in the Republic of Kosova-a feat that had not yet been accomplished in this newly independent (2008) eastern European country. Three Holstein heifers at the Iowa State University dairy farm were superovulated with a conventional descending dose regimen of FSH (Folltropin). Approximately 12 and 24h after the observed onset of oestrus, heifers were inseminated with semen from a single Red Holstein bull. Embryos were non-surgically collected and washed in accordance with IETS procedures for sanitary handling of embryos. Embryos were cryopreserved for subsequent direct transfer. After obtaining an import permit from the Kosovo Food and Veterinary Agency, embryos were approved for export to the Republic of Kosova by the US Department of Agriculture, Animal Plant Health Inspection Service. Embryos were shipped via an express courier service. A total of 19 embryos were received in the Republic of Kosova. Recipients were monitored for signs of naturally occurring oestrus, and immediately before transfer, embryos were thawed by holding in air for 3 to 5s followed by placement into a 37°C water bath for 25 to 30s. The first-ever bovine embryo transfer calf in the Republic of Kosova was born July 6, 2015. A total of 9 calves were born from the 19 embryos transferred (47.4% embryo survival rate). Results of this applied field study show that bovine embryo transfer is feasible in the Republic of Kosova. Embryo transfer will be used to improve the quality of dairy cattle genetics in the Republic of Kosova and to subsequently increase the national supply of milk, decrease dependence on milk imports, and increase food security of the nation.

Tecnologías Reproductivas Modernas para Mejorar la Producción Pecuaria

Cattle contribute significantly to the global supply of animal-derived proteins which are an important part of a well-balanced human diet. With the human population increasing by 1 billion people every 13 years, there will be an estimated 9.6 billion people on planet Earth by the year 2050. To attain global food security the amount of available food will need to double between now and then. Enhancing reproductive efficiency is a prerequisite for boosting production of meat and milk from cattle, and, fortunately, an arsenal of modern reproductive technologies is available to assist with that effort. The objective of this manuscript is to provide an overview of reproductive biotechnologies that can bolster the efficient production of meat and milk from cattle. Protocols for synchronization of estrus and synchronization of ovulation facilitate more efficient artificial insemination using conventional or sexsorted semen. In vivo and in vitro production of preimplantation embryos from genetically superior females enable creation of multiple offspring with high production potential. Biochemical and ultrasonographic methods for pregnancy testing identify non-pregnant females that can be re-mated or sold to prevent wastage of valuable feed resources. Somatic cell nuclear transfer is used to create copies of highly productive animals, and genome editing of zygotes provides a novel opportunity to selectively enhance the genetic makeup of cattle for the benefit of animal and human health. Wise use of these reproductive technologies will increase food production from cattle and will help alleviate world hunger.

Ovarian follicular dynamics in purebred and crossbred Boran cows in Ethiopia

Boran is an endangered breed of cattle indigenous to Ethiopia and the relatively poor understanding of its reproductive physiology has impeded efforts to maximize reproductive performance of the breed. This study characterized ovarian follicular dynamics in 9 purebred Boran and 8 Boran×Holstein (B×H) crossbred cows. Ovaries of all 17 cows were examined once per day for 61 consecutive days (encompassing three periods of estrus) using transrectal ultrasonography. The mean (±standard error of mean) inter-ovulatory interval (IOI) was similar (P>0.05) in Boran (19.4 ± 0.2 days) and B×H cows (20.1 ± 0.4 days). Two (in 79% of estrous cycles) or three (in 21% of cycles) follicular waves per IOI were observed and IOI was shorter (P 0.10) in both genotypes (15.8 ± 1.5 mm in Boran and 19.4 ± 2.9 mm in B×H). Boran cows possessed a greater (P<0.001) total number of ovarian follicles than B×H cows and both genotypes displayed more (P<0.05) activity on their right than left ovary. Results of our study have provided novel insights into the normal reproductive physiology of the Boran breed. Key words: Boran, estrous cycle length, follicular dynamics, follicular waves, dominant follicle.