Juvenile invitro embryo transfer (JIVET) is an assisted reproductive technology (ART) with the potential to produce numerous offspring from a single young female goat at 4 to 8 weeks of age. It has been reported in small ruminants that there can be a marked variable response to the administration of exogenous hormones for superovulation, the subsequent number of oocytes generated, and subsequent embryo developmental potential. The industry standard (as well as the recommendation of commercial media suppliers) invitro maturation time is 21 to 24h for conventionally derived oocytes. This study investigated multiple maturation times for JIVET-derived oocytes: 16, 22, and 28h. Oocytes were collected from four JIVET animals at 6 to 8 weeks of age. The hormonal superovulation regimen used on the juvenile animals consisted of 4×40-mg FSH injections at ∼12h apart and a 400IU of PMSG injection given with the first FSH injection. Surgical recovery of the oocytes via a midline laparotomy was performed the day following the last FSH injection. All of the oocytes were collected via aspirating follicles that were 4mm and larger. Oocytes with compact cumulus cells subsequently underwent IVM, IVF, and invitro culture (IVC) utilising IVF Bioscience media and methods. A single straw of identical cryopreserved/thawed semen from the same buck was utilised for each of the IVF procedures. The results were (37/88) 42%, (37/85) 44%, and (39/91) 43% cleaved and (23/88) 26%, (24/85) 28%, and (28/91) 31% blastocyst rate based on respective maturation times for JIVET-derived ova. Development rate during the cleavage stage and blastocyst stage was analysed using a repeated-measures logistic regression model utilising generalized estimating equations (GEE), with maturation time as fixed effect and a compound symmetry within subject (juvenile goat) covariance structure. The main effect of maturation time on the odds of development during the cleavage stage (P=0.8727) and blastocyst stage (P=0.3857) was not significant. These results indicate that the time in maturation media does not have as profound an effect on development to blastocysts as a factor in the variability reported by other laboratories. The development rate of embryos from one juvenile goat produced very high blastocyst rates of (5/12) 42%, (11/12) 92%, and (11/15) 73%, respectively. Additional logistic regression analysis showed that the odds of development in this juvenile donor was significantly different compared with the other donors (pooled) during the cleavage stage at 16h (P=0.0083) and 28h (P=0.0021) maturation times. Likewise, the odds of development in this donor was significantly different than that of the other donors (pooled) during the blastocyst stage at 22h (P=0.0002) and 28h (P=0.0003) maturation times. This further indicates the wide variation of oocyte quality from JIVET-derived oocytes and indicates potential for higher development rates at 22 and 28h in this specific goat.

At LFB USA, Inc., transgenic goats are utilized for the production of recombinant human protein thera-peutics in their milk through the rPROTM Technology platform. This retrospective analysis and report describes the results of induced parturition and its use as a management tool in this large herd of dairy goats. Over a three-year period, 342 does received pronuclear microinjected (MI) embryos transferred into the oviductal lumen via midline laparotomy (day 1). To initiate the induction process, does were given intramuscular injections (IM) of 10 mg each of prostaglandin (Lutalyse (R)) and dexamethasone to induce parturition on days 144-148 of pregnancy. Mean and Standard Deviation (+/- SD) time to partu-rition was 36.7 (+/- 6.5) hours. Does were given these injections at 4pm on Sundays with an expected kidding time of late Monday into Tuesday morning. Of the 342 does, 333 or 97% had kidded by 3pm the following Tuesday, and 313 or 91% kidded in the 18 h between 9pm Monday and 3pm on Tuesday or between 29 and 47 h post induction. By the end of Tuesday, most kids had received colostrum and were transferred to the nursery. The incidences of kid mortality and retained placenta were 2.5% and 1.5%, respectively, clearly achieving a priority at this commercial operation for generating a high percentage of live kids (97.5%) of marked value being produced. The use of induced parturition allowed this large dairy operation to designate two 9-h time blocks in which to concentrate parturition times within the herd. This facilitated strategic scheduling to optimize availability of staff, in order to assist with parturition, separate kids from the dam at birth, and ensure adequate and prompt feeding of colostrum. Predicting the time of kidding in this way can serve as an effective management tool, especially to help reduce kid mortality and prevent disease spread by restricting suckling of colostrum. (c) 2021 Elsevier Inc. All rights reserved.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

The objective of this study was to investigate a method of oviducal semen deposition as a strategy for producing offspring from poor-quality cryopreserved goat sperm. Invitro fertilisation (IVF) and AI are common assisted reproductive technologies used in small ruminants, but they have varied results in the goat. The use of poor-quality cryopreserved-thawed sperm (<50% live/dead ratio at post-thaw) can decrease the rate of success. These procedures were performed in the month of November in Central Massachusetts in the United States (42° N). Seven 10-year-old dairy goats (Saanen, Toggenburg, and Alpine breeds) were synchronised and superovulated using a progesterone implant on Day 0, a prostaglandin injection at Day 7, two daily injections of 36mg of FSH ~12h apart on Days 12-15, and progesterone implant removal on Day 14 followed by an injection of 50µg of gonadotrophin-releasing hormone. Sperm deposition was performed on Day 17 (72 h after implant removal). The animals were anaesthetised using a standardised protocol, intubated, and maintained using isoflurane, and sterile prep was performed before a midline laparotomy procedure. Straws from a single ejaculate from a transgenic founder that was cryopreserved using a commercial two-step glycerol-egg yolk-based extender were used. A straw from this collection was post-thawed 30 days after collection and, using a commercial live/dead stain, 67% live sperm was determined. The optimal type of sperm prep and sperm concentration is unknown and may be dependent on sperm quality. Therefore, different gradient preps using Vitrolife SpermGrad at three volumes (1.5 (used on two animals), 1.0, and 0.5mL) as well as two volumes of IVF Bioscience Bovine BO-SemenPrep (4.0mL (used on two animals) and 2.0mL) were used. All five pellets were diluted in 1.0mL of IVF Bioscience Bovine BO-IVF media. Sperm concentrations ranging from 75×106 to 27×106 spermmL−1 were deposited into one oviduct; then, a 10:1 dilution was performed and 7.5×106 to 2.7×10 spermmL−1 were deposited into the contralateral oviduct. The depositions were performed just proximal to the uterotubal junction in a volume of 0.1mL of diluent via a tuberculin syringe attached to a 20-gauge needle. Two days following the procedure, oviducts were flushed postmortem from three of the seven randomly selected goats. All three had fertilised embryos, and nineteen 8-cell embryos were retrieved. Three of these embryos were surgically transferred to the distal uterine horn of a suitable recipient. The recipient became pregnant and produced a single offspring. The remaining four of seven goats were killed 41 days post-surgery. Two of the four goats were pregnant, with one carrying one fetus and the other carrying five fetuses. Further studies are needed to optimise this method, but these initial results indicate that oviducal semen deposition directly into the oviduct proximal to the uterotubal junction may be a suitable alternative for producing offspring from suboptimal cryopreserved-thawed goat sperm.

At LFB USA, Inc., the ultimate use for transgenic cloned goats is for the production of recombinant human protein therapeutics in their milk. This retrospective analysis of the Somatic Cell Nuclear Transfer (SCNT) program, spanning from 1998 to 2010, examined parameters potentially affecting the outcomes and efficiencies in this commercial operation. Over 37,000 + ova were utilized in the SCNT protocol producing a total of 203 cloned goats. Fifty one (51) clones were produced from non-transfected (transgenic and non-transgenic animal donor) cell lines and 152 clones were produced from transfected cell lines. Comparisons and summaries of (a) transfected versus non-transfected cell lines, (b) relationship of SCNT parameters to offspring produced, (c) skin versus fetal cells, (d) fresh versus cryopreserved cells, (e) parameters from all cell lines used versus those producing SCNT offspring, (f) variation among cell sources, (g) methods of SCNT parturition management and effects on live offspring, and lastly (h) SCNT variation by program are reported. Findings indicate that (a) non-transfected cell lines were more efficient versus transfected cell lines in generating viable cloned offspring on a per reconstructed embryo transferred basis, (b) transfected fetal fibroblasts had improved efficiency versus transfected skin fibroblasts, (c) the percentage of non-transfected cell lines that produced offspring was statistically higher than transfected cell lines, (d) and induction of parturition improved the percentage of viable offspring. In summary, this retrospective analysis on the SCNT process has identified certain parameters for improved efficiency in producing viable cloned goats in a commercial setting.

The production of transgenic founder dairy goats (cross-bred Saanens, Alpines, Toggenburgs, and Nubians) involves the collection, microinjection, and transfer of numerous embryos into suitable recipient goats to ultimately produce a transgenic founder(s). The objective of this study was to determine the most efficient number of microinjection embryos to transfer to suitable recipients for transgenic founder generation. This is critically important in a commercial production program, as it impacts the goal for the number of embryos collected from donors, number of recipients utilised, and, hence, the overall number of surgical procedures being performed. The entire embryo collection, transfer, and founder-generation process is continuously being evaluated for ways to become more efficient in producing transgenic animals. During LFB USA’s commercial founder-production campaigns over the years (1997-2017), pronuclear microinjection was performed and 3, 4, or 5 embryos were transferred to female goat recipients. The recipients were synchronized using a progesterone implant on Day 0, a prostaglandin injection at Day 7, an injection of 300-500IU of pregnant mare serum gonadotropin on Day 13, progesterone implant removal on Day 14, and surgical transfer of pronuclear microinjected 1- or 2-cell embryos into the oviduct on Day 17. The individual totals and calculation for offspring per embryos transferred was compared for 3, 4, and 5 embryos transferred per recipient and was determined to be (1659/8637) 0.19, (912/4548) 0.20, and (112/675) 0.17, respectively. These embryo efficacy ratios were not significantly different (P>0.05) using the Wald Chi-squared test under logistic regression, and suggests that the number of offspring born is not impacted by number of embryos transferred. Seasonality was also evaluated in this production environment located in North America, with in-season being considered September to December and out-of-season being January to July. Nulliparous recipients during in-season (September to December) embryo transfer operations produced a significant difference, with totals and calculation for (offspring per embryo transferred) of (470/2346) 0.20, (260/1088) 0.24, and (23/190) 0.12 for 3, 4, and 5 embryo transfers, respectively (Table 1). This data indicates that when using nulliparous recipients during the in-season, transferring 4 embryos is optimal for offspring produced. Table 1.Comparison of the individual totals and the calculation of (offspring/embryo) by parity and season

Superovulation of donor animals is essential in the production of transgenic founder goats generated through microinjection. There can be a marked variable response to the exogenous hormones used for superovulation. The objective of this study was to examine how the superovulatory response of individual goats affected the ability of the fertilized, microinjected embryos to develop into offspring. The donors were superovulated using a progesterone implant on Day 0, a prostaglandin injection at Day 7, 2 injections ~12 h apart of 32 to 36 mg of FSH on Day 12 to 15, progesterone implant removal on Day 14, bred by intact bucks several times starting on Day 15 to 16, an injection of 50 μg of gonadotropin-releasing hormone, and surgical collection of 1- to 2-cell embryos from retrograde flushing of the oviduct on Day 17 (~24-48 h, 1-2 days after breeding). Surgical collection allows for an accurate ovulation point (OP) count before the oviduct being retrograde flushed and ova collected and counted. Data from donor animals were grouped by superovulatory response based on OP counts of 1-10, 11-20, 21-30, or u003e30. The number of donors that contributed per group were 130, 280, 175, and 52, respectively. The recovery rate was 76, 72, 68, and 62%, respectively. After collection, ova were viewed under a dissecting microscope and assessed for fertilization by identifying pronuclei, and 1 pronucleus was microinjected. The fertilization rate was 47, 52, 51, and 56%, respectively. The survivability rate after microinjection was 80, 76, 75, and 76%, respectively. Surviving embryos were transferred (3-5) into recipient goats following a 2- to 6-h in vitro culture (as 1- to 2-cell embryos), allowing for a suitable period to assess viability post-injection. Further in vitro development rates were not assessed because of the short timeframe the embryos stayed in culture. The conception rates were 71, 56, 65, and 53%, respectively, and abortion rates were 23, 10, 14, and 9%, respectively. As some recipients received embryos from multiple donors, this data could not be included in the analysis as identifying which offspring were from the corresponding embryo group could not be confirmed. Data were analysed using SAS software (version 9.4, SAS Institute Inc., Cary, NC, USA). The Wald chi square test under linear regression was used to analyse the number of offspring produced per embryo transferred. No significant differences were found between groups (all P-values were u003e 0.05). This analysis indicated that the range of superovulation response does not affect the developmental competence of the pronuclear microinjected embryo or the ability to produce viable offspring. Table 1.Comparison of the donor ovulation counts, number of embryos transferred, offspring produced and overall efficiency

•Caprine herpesvirus 1: a successful eradication program in a dairy goat herd.•Serum neutralization assay was a useful screening test for caprine herpesvirus 1.•Seropositive goats mostly remained subclinical.•Seropositive bucks were unlikely to spread disease to does during breeding.•Eradication of disease in a herd is achievable with proper screening and herd management.

Production of transgenic founder goats involves introducing and stably integrating an engineered piece of DNA into the genome of the animal. At LFB USA, the ultimate use of these transgenic goats is for the production of recombinant human protein therapeutics in the milk of these dairy animals. The transgene or construct typically links a milk protein specific promoter sequence, the coding sequence for the gene of interest, and the necessary downstream regulatory sequences thereby directing expression of the recombinant protein in the milk during the lactation period. Over the time period indicated (1995–2012), pronuclear microinjection was used in a number of programs to insert transgenes into 18,120, 1- or 2- cell stage fertilized embryos. These embryos were transferred into 4180 synchronized recipient females with 1934 (47%) recipients becoming pregnant, 2594 offspring generated, and a 109 (4.2%) of those offspring determined to be transgenic. Even with new and improving genome editing tools now available, pronuclear microinjection is still the predominant and proven technology used in this commercial setting supporting regulatory filings and market authorizations when producing founder transgenic animals with large transgenes (> 10 kb) such as those necessary for directing monoclonal antibody production in milk.

To the Editor: In 1999, we reported the production of the world's first cloned goats by somatic cell nuclear transfer (SCNT)1. This came after reports of the first cloned sheep, cow and mouse and was followed by cloning of many other large animal species2. We report here that these three cloned… Download references

Germline stem cells (GSCs) can be used for large animal transgenesis, in which GSCs that are genetically manipulated in vitro are transplanted into a recipient testis to generate donor‐derived transgenic sperm. The objectives of this study were to explore a non‐viral approach for transgene delivery into goat GSCs and to investigate the efficiency of nucleofection in producing transgenic sperm. Four recipient goats received fractionated irradiation at 8 weeks of age to deplete endogenous GSCs. Germ cell transplantations were performed 8–9 weeks post‐irradiation. Donor cells were collected from testes of 9‐week‐old goats, enriched for GSCs by Staput velocity sedimentation, and transfected by nucleofection with a transgene construct harboring the human growth hormone gene under the control of the goat beta‐casein promoter (GBC) and a chicken beta‐globin insulator (CBGI) sequence upstream of the promoter. For each recipient, transfected cells from 10 nucleofection reactions were pooled, mixed with non‐transfected cells to a total of 1.5 × 108 cells in 3 ml, and transplanted into one testis (n = 4 recipients) by ultrasound‐guided cannulation of the rete testis. The second testis of each recipient was removed. Semen was collected, starting at 9 months after transplantation, for a period of over a year (a total of 62 ejaculates from four recipients). Nested genomic PCR for hGH and CBGI sequences demonstrated that 31.3% ± 12.6% of ejaculates were positive for both hGH and CBGI. This study provides proof‐of‐concept that non‐viral transfection (nucleofection) of primary goat germ cells followed by germ cell transplantation results in transgene transmission to sperm in recipient goats. Mol. Reprod. Dev. 79: 255–261, 2012. © 2011 Wiley Periodicals, Inc.

Transgenic dairy goats expressing recombinant molecules in their milk have been validated as a viable method for producing human therapeutic proteins. Although maintaining a closed herd ensures biosecurity within a facility, the ability to introduce new genetics into the herd can be difficult. In this work we determined the ability to use cryopreserved caprine semen, imported from New Zealand into the United States, for IVF as a method to increase the genetic diversity of the GTC Biotherapeutics closed caprine herd. Semen was collected from bucks owned by GTC Biotherapeutics and maintained in New Zealand. The bucks were serologically screened for goat pathogens prior to collection, and were maintained in quarantine during semen collection. Separate single experiments were performed using cryopreserved semen from each of 2 different bucks (NZ1 and NZ2). One or 2 straws of semen (107/0.25 mL straw) from each buck were thawed and then purified using a Percoll gradient. Ovulated oocytes surgically collected from superovulated does were co-incubated with sperm (5 × 105 mL–1) in Brackett-Oliphant medium supplemented with 10% fetal bovine serum, 7.7 mm calcium lactate plus 2.5 μg mL–1 of heparin for 18 h at 38°C. Presumptive zygotes were transferred to equilibrated SOF plus 0.8% BSA and cultured in vitro for 24 h. On Day 2 cleavage was determined and, as an added precaution, embryos selected for transfer were washed per the IETS protocol for the sanitary handling of embryos. Five 2-cell to 8-cell embryos from individual donors were surgically transferred to a single oviduct of each synchronized surrogate recipient. Pregnancies were determined by ultrasonography. Pregnancy rates for recipients at Day 50 of gestation (71 v. 67% pregnant), at term (100 v. 100%), and the proportion of offspring born from total embryos transferred (17 v. 23% offspring) were comparable for buck NZ1 and buck NZ2, respectively (P > 0.05). A total of 13 offspring (6 bucks and 7 does) were produced from 9 different oocyte donors. These results demonstrate that cryopreserved caprine semen, imported from New Zealand into the United States, can be used for IVF to introduce new genetics into a closed biosecure caprine herd. The use of IVF, compared with AI, allows more offspring to be produced per straw of semen. In addition, IVF offers the advantage of accelerated genetic gain by producing multiple offspring from elite does with more desirable lactation, reproduction, and conformation traits. Beyond the new F1 animals produced by IVF, several techniques (natural mating, AI, or IVF) can then be used to quickly disseminate the new genetics into both the nontransgenic and transgenic herds. Finally, skin cells obtained from the female IVF offspring or fetal cells derived from any pregnancies of the initial IVF offspring could also be used to generate transfected cells as karyoplast donors for future somatic cell nuclear transfer work. Table 1. Summary of caprine IVF

We explored whether exposure of mammalian germ line stem cells to adeno-associated virus (AAV), a gene therapy vector, would lead to stable transduction and transgene transmission. Mouse germ cells harvested from experimentally induced cryptorchid donor testes were exposed in vitro to AAV vectors carrying a GFP transgene and transplanted to germ cell-depleted syngeneic recipient testes, resulting in colonization of the recipient testes by transgenic donor cells. Mating of recipient males to wild-type females yielded 10% transgenic offspring. To broaden the approach to nonrodent species, AAV-transduced germ cells from goats were transplanted to recipient males in which endogenous germ cells had been depleted by fractionated testicular irradiation. Transgenic germ cells colonized recipient testes and produced transgenic sperm. When semen was used for in vitro fertilization (IVF), 10% of embryos were transgenic. Here, we report for the first time that AAV-mediated transduction of mammalian germ cells leads to transmission of the transgene through the male germ line. Equally important, this is also the first report of transgenesis via germ cell transplantation in a nonrodent species, a promising approach to generate transgenic large animal models for biomedical research.

The efficiency of germ cell transplantation, the procedure of transferring germ cells from a donor male into the testes of recipient males, can be greatly increased by reduction of endogenous germ cells in recipient animals. To develop effective methods for suppression of endogenous spermatogenesis in potential pig and goat recipients, we either administered busulfan to pregnant sows or irradiated the testes of immature goats. Piglets from sows treated twice with busulfan (7.5 mg/kg) at days 98 and 108 of gestation showed reduced gonocyte numbers at 2, 4, and 8 weeks of age and reduced initiation of spermatogenesis at 16 weeks of age. For goats, groups of 3 kids at 1, 5, or 9.5 weeks of age received fractionated irradiation of the testes with 3 doses of 2 Gy on 3 consecutive days. At 2 months after irradiation, 5%-10% of seminiferous tubule cross sections contained pachytene spermatocytes, compared with 50%-100% in controls. At 3 months after irradiation, spermatozoa appeared in 20% of tubule cross sections in all treated goats and in 100% of tubules in control goats. By 6 months after irradiation, spermatogenesis had recovered in 60% of tubules in goats treated at 5 or 9.5 weeks of age but in only 29% of tubules after treatment at 1 week of age. Therefore, late gestation in utero treatment of pigs with low doses of busulfan and testicular irradiation of goats at 1 week of age will result in a reduction in the endogenous germ cell population that could facilitate donor cell colonization after germ cell transplantation.

No information is available concerning how the maturation environment controls the metabolism of goat oocytes. The objectives of this experiment were to: (1) Determine the concentrations of glucose, lactate, and pyruvate in caprine follicular fluid; and (2) Investigate the effects of physiological concentrations of glucose and lactate in the in vitro maturation (IVM) medium on the metabolism (glycolysis and pyruvate oxidation), protein content, and developmental competence of caprine oocytes and cumulus-oocyte complexes (COCs). Abattoir-derived COCs were matured for 18-20 hr in a defined, SOF-based medium containing 0.75, 1.5 (follicular fluid = 1.4 mM), or 3.0 mM glucose, and 3.0, 6.0 (follicular fluid = 7.1 mM), or 12.0 mM L-lactate. The protein content of oocytes and COCs was not affected (P > 0.05) by the concentration of glucose and lactate in the maturation medium. Increasing glucose and lactate decreased (P < or = 0.05) glycolytic activity of oocytes, without affecting (P > 0.05) pyruvate oxidation. In COCs, increasing glucose concentrations tended (P = 0.07) to decrease glycolysis. When metabolic activity was corrected for protein content (pmol/microg protein/3 hr), increasing glucose or lactate concentrations in the medium decreased (P < or = 0.05) pyruvate oxidation in oocytes, but increased (P < or = 0.05) pyruvate oxidation in COCs. Embryonic development (cleavage and blastocyst development, hatching, and cell number) was not affected (P > 0.05) by the glucose and lactate concentrations tested. These results indicate that concentrations of glucose and lactate in the medium have cell type-specific effects on metabolism of oocytes and COCs, but do not affect developmental competence within the range of concentrations tested.

The production of recombinant proteins in the milk of transgenic animals is an alternative to traditional cell culture methodology. Transgenic rabbits can serve in the small-scale production of recombinant proteins, underscoring the need to maintain valuable transgenic lines. In this study, the authors used cryopreserved transgenic rabbit semen to artificially inseminate does, demonstrating the utility of this method for the reestablishment of a transgenic rabbit herd.