Alpacas are increasingly incorporated into reproductive and biomedical research due to distinctive physiological and immunological characteristics that extend their relevance beyond agricultural applications. As camelids, alpacas exhibit induced ovulation, prolonged follicular dynamics, asymmetric uterine physiology, and a unique immune system characterized by heavy-chain-only antibodies. These features support their use in comparative and translational research, particularly in reproductive biology and genetic engineering. Laparoscopic ovum pick-up (LOPU) is a minimally invasive approach for oocyte recovery in species with complex reproductive anatomy, allowing direct ovarian visualization, precise follicular aspiration, and reduced tissue trauma compared with transvaginal techniques. The objective of this study was to develop and standardize a reproducible in vivo oocyte retrieval protocol in alpacas using LOPU. Eight adult female alpacas (3-8 years of age) were enrolled in this study and subjected to repeated LOPU sessions conducted once per month throughout the study period. Alpacas were evaluated by transrectal ultrasonography, and animals presenting ovarian follicles ≥7 mm received 50 µg gonadotropin-releasing hormone intramuscularly. Ovarian superstimulation was induced using a total dose of 200 mg follicle-stimulating hormone administered in a decreasing twice-daily regimen over four days. LOPU was performed 12-14 h after the final hormone administration under general anesthesia induced with ketamine, xylazine, and butorphanol and maintained with isoflurane. A three-port laparoscopic approach with carbon dioxide insufflation was used. Follicles were aspirated using a 20-gauge needle connected to a regulated vacuum system (25-30 mmHg; 13-15 mL/min). Oocytes were recovered in TCM-199 medium and evaluated under a stereomicroscope. Alpacas exhibited a mean of 14.88 ± 3.85 follicles, with 10.94 ± 3.62 follicles aspirated per session and 6.68 ± 2.21 oocytes recovered, corresponding to a recovery rate of 61.30 ± 8.21%. These findings suggest that LOPU may represent a viable approach for in vivo oocyte retrieval in alpacas, with potential applications in reproductive biotechnology workflows.

Mammalian somatic cell nuclear transfer (SCNT) is widely used in agricultural research and biomedicine, but remains inefficient and requires large number of oocytes, typically obtained from slaughterhouses (SH) or cull animals with or without superstimulation (Culls-FSH and Culls). Laparoscopic ovum pick-up (LOPU) is a less invasive method to collect oocytes and improves SCNT embryo development. Yet no study has directly compared all four sources of oocytes on SCNT embryo development in sheep. This study evaluated the effect of oocyte source on in vitro maturation (IVM) and SCNT embryo in vitro and in vivo development. IVM rates were significantly higher in LOPU-derived oocytes than SH oocytes both in the breeding season (93.6% vs 71.9%, P=0.0007) and non-breeding season (91.1% vs 66.5%, P=0.0013). In the breeding season, the LOPU group produced a higher SCNT blastocyst rate than the SH group (41.7% vs 11.8%, P=0.045), whereas no difference was observed in the non-breeding season. Because higher blastocyst development was expected, fewer one-cell embryos were transferred per recipient for the LOPU group compared with SH, Culls-FSH, and Culls (4.4 vs. 15.8, 14.5, and 18.5, P < 0.001). Neither the initial pregnancy rates nor the full-term rates differed among groups. But LOPU group showed a significantly greater SCNT efficiency than the SH and two Culls groups (14.0% vs 1.94%, 1.21%, and 1.97%, P < 0.001). In conclusion, LOPU produced the highest-quality oocytes among the four oocyte sources, resulting in greater oocyte maturation and more developmentally competent embryos both in vitro and in vivo, despite limited number of embryo transfers (n = 5).

Somatic cell nuclear transfer (SCNT) is one of the primary methods for production of genetically engineered sheep, which allows for gene editing or transgene introduction in somatic cells. The use of SCNT eliminates the risk of genetic mosaicism in embryos and animals that is commonly observed after zygote micromanipulations. This retrospective analysis of SCNT in sheep performed at Utah State University, spanning from 2016 to 2021, examined parameters that may impact pregnancy and full-term development, including donor oocytes (donor age), donor cell lines, SCNT parameters (time of oocyte activation following SCNT, number of transferred embryos, in vitro maturation and culture conditions), and recipients (surgical number and ovulatory status), as well as factors that may correlate with large offspring syndrome or abnormal offspring syndrome (LOS/AOS) in the fetuses and lambs. Our findings indicated that compared to prepubertal oocytes, the SCNT embryos produced from adult sheep oocytes had comparable in vitro maturation rates, pregnancy and full-term development rates, as well as SCNT efficiency. In addition, earlier activation time of SCNT embryos (e.g. 24-26 h post maturation) was correlated to the early pregnancy loss rate, full-term rate, and SCNT efficiency. Compared to our standard serum-containing medium, commercial serum-free culture medium showed a positive correlation with the full-term development of sheep SCNT embryos. Transferring 15-30 embryos per recipient resulted in consistently good pregnancy rates. Surgical numbers and ovulatory status (having at least one follicle between 6 and 12 mm in size or a corpus hemorrhagicum (CH)) of recipients did not affect pregnancy and full-term development rates. In summary, this retrospective analysis identified parameters for improving pregnancy and full-term development of SCNT embryos in sheep.

CONTEXT:In vitro maturation is an important process in the production of embryos. It has been shown that three cytokines, fibroblast growth factor 2, leukemia inhibitory factor and insulin-like growth factor 1 (FLI), increased efficiency of in vitro maturation, somatic cell nuclear transfer (SCNT) blastocyst production, and in vivo development of genetically engineered piglets.AIMS:Assess effects of FLI on oocyte maturation, quality of oocytes, and embryo development in bovine in vitro fertilisation (IVF) and SCNT.KEY RESULTS:Cytokine supplementation resulted in significant increases in maturation rates and decreased levels of reactive oxygen species. Oocytes matured in FLI had increased blastocyst rates when used in IVF (35.6%vs 27.3%, P <0.05) and SCNT (40.6%vs 25.7%, P <0.05). SCNT blastocysts contained significantly more inner cell mass and trophectodermal cells when compared to the control group. Importantly, SCNT embryos derived from oocytes matured in FLI medium resulted in a four-fold increase in full-term development compared to control medium (23.3%vs 5.3%, P <0.05). Relative mRNA expression analysis of 37 genes associated with embryonic and fetal development revealed one gene had differential transcript abundance in metaphase II oocytes, nine genes at the 8-cell stage, 10 genes at the blastocyst stage in IVF embryos and four genes at the blastocyst stage in SCNT embryos.CONCLUSIONS:Cytokine supplementation increased efficiency of in vitro production of IVF and SCNT embryos and in vivo development of SCNT embryos to term.IMPLICATIONS:Cytokine supplementation is beneficial to embryo culture systems, which may shed light on requirements of early embryo development.

Many breeds of dairy cattle exist, but questions remain about their relative performance within exclusively forage-based systems in the United States. This paucity of data is especially apparent in relation to heifer development, which is the second largest expense on most dairy farms. We evaluated the performance of prepubertal heifers from four different genetic backgrounds (“breeds”) within a rotational grazing system in the Intermountain West. For each of two 105-day grazing seasons, 24 dairy heifers from each of four different breeds [Holstein (HO), Jersey (JE), Holstein/Jersey crossbred (HJ), and Swedish Red/Holstein/Montbeliarde crossbred (SHM)] were randomly assigned to one of two pasture treatments that consisted of either grass only (MONO) or grass interseeded with the legume birdsfoot trefoil (BFT; MIX). Main effects of treatment and breed (and potential interactions) on heifer average daily gain (ADG), change in percent mature body weight (CPMBW), fecal egg count (FEC), rumen volatile fatty acids (VFA), and body condition scores (BCS) were evaluated. There was a significant effect of pasture treatment on ADG, with MIX heifers averaging 0.48 kg of gain/day, whereas MONO heifers averaged 0.29 kg/day (P < 0.0001). Change in BCS was significantly affected by treatment (P = 0.0012) and breed (P < 0.0001). Significant effects of treatment (P < 0.0001), breed (P = 0.0005), and treatment*breed (P = 0.0005) on CPMBW were also found. Treatment and breed had no effect on FEC or total rumen VFA content (P > 0.05). Overall, we found that Jerseys were able to gain a higher percentage of their mature body weight and lose less body condition while on pasture than heifers of other breeds. We also found that grazing BFT can have significant positive effects on ADG, BCS, and CPMBW. Further research is needed to more thoroughly evaluate the influence of heifer genetics (breed) on metrics of growth, health, reproductive performance, and production in pasture settings.

This study investigates the effects that different forages in a rotational grazing system have on pH and VFA proportions of rumen fluid in organically raised dairy heifers. In 2018, 81 yearling Jersey heifers were assigned to one of nine treatments, including a conventional dry lot control (TMR) or one of eight pasture treatments. Pasture treatments included: tall fescue (TF), meadow bromegrass (MB), orchard grass (OG), perennial ryegrass (PR) and each individual grass interseeded with birdsfoot trefoil (BFT). Every 35-d, over a 105-d period, rumen fluid was collected using an oral stomach tube. Immediately following collection, pH was measured and recorded. Rumen fluid collected for VFA analysis was stabilized with acid and placed on wet ice and eventually frozen at -80° C until further analysis. Repeated measures analyses were performed using PROC MIXED in SAS (version 9.4) to determine how treatment affected each measurement over the total 105-day period. Time had a significant effect (P < 0.001) on pH ofrumen fluid over the 105-d growth period. Additionally, treatment had a significant effect (P < 0.001) on pH of rumen over the 105-d growth period. Animals that consumed the TMR diet had decreased rumen pH compared to other treatments. The proportions of acetate, propionate, isobutyrate, butyrate, isovalerate, and valerate were all significantly affected (P < 0.001) by time over the 105-d growth period. Treatment had an effect (P < 0.05) on the proportions ofpropionate and butyrate and had a tendency (P = 0.06) to affect the proportion of isovalerate. In general, animals on the TF pastures had decreased (P < 0.05) proportions of these VFAs in the rumen fluid. The proportions of acetate, isobutyrate, and valerate were not affected (P < 0.05) by treatment. These data provide insight into how different forages in organic pastures affect rumen characteristics of growing Jersey heifers.

Dairy heifers developed in certified organic programs, especially those utilizing pasture-based management schemes, have lower rates of gain than heifers raised in nonorganic confinement production systems in temperate climates, such as in the Intermountain West region of the United States. This study investigates the effects that different forages in a rotational grazing system have on development of organically raised Jersey heifers. Over 3 years, 210 yearling Jersey heifers were randomly assigned to one of 9 treatments, including a conventional confinement control where animals were fed a total mixed ration or one of 8 pasture treatments: Cache Meadow bromegrass (Bromus riparius Rehmann), QuickDraw orchard grass (Dactylis glomerata L.), Amazon perennial ryegrass (Lolium perenne L.), or Fawn tall fescue (Schendonorus arundinaceus [Schreb.] Dumort) and each individual grass interseeded with birdsfoot trefoil (Lotus corniculatus L., BFT). Each treatment had 3 blocks/yr over the 3-yr period, with each block having a 0.4 ha pasture of each treatment. Every 35 d, over a 105-d period, heifers were weighed and measured for hip height, and blood samples were collected to determine serum insulin-like growth factor-1 and blood urea nitrogen concentrations. Fecal egg counts were also assessed. Heifer body weight (BW), blood urea nitrogen, and insulin-like growth factor-1 concentrations were affected by treatment when analyzed over time. Heifers on grass-BFT pastures had increased BW compared with heifers on monoculture grass pastures. Heifers receiving a total mixed ration or perennial ryegrass+BFT had increased BW gain over the 105-d period compared with heifers grazing tall fescue+BFT, orchard grass, perennial ryegrass, meadow bromegrass, or tall fescue. Individually for all grass species, heifers grazing +BFT pastures had greater ending BW and weight gain than heifers grazing the respective grass monocultures. Furthermore, weight gain for heifers on perennial ryegrass+BFT, meadow bromegrass+BFT, and orchard grass+BFT were not different from those on a total mixed ration. Heifers grazing grass-BFT pastures had increased blood urea nitrogen compared with heifers grazing monoculture grass pastures. Heifer hip height and fecal egg counts were not affected by treatment. These results show that the addition of BFT to organic pasture improves growth of grazing replacement heifers. Economic analyses also demonstrate that interseeding grass pastures with BFT results in an increased economic return compared with grazing monoculture grass pastures. Grass pastures interseeded with BFT may be a sustainable option to achieve adequate growth of Jersey heifers raised in an organic pasture scenario in a temperate climate.

To address the unmet needs for human polyclonal antibodies both as therapeutics and diagnostic reagents, building upon our previously established transchromosomic (Tc) cattle platform, we report herein the development of a Tc goat system expressing human polyclonal antibodies in their sera. In the Tc goat system, a human artificial chromosome (HAC) comprising the entire human immunoglobulin (Ig) gene repertoire in the germline configuration was introduced into the genetic makeup of the domestic goat. We achieved this by transferring the HAC into goat fetal fibroblast cells followed by somatic cell nuclear transfer for Tc goat production. Gene and protein expression analyses in the peripheral blood mononuclear cells (PBMC) and the sera, respectively, of Tc caprine demonstrated the successful expression of human Ig genes and antibodies. Furthermore, immunization of Tc caprine with inactivated influenza A (H7N9) viruses followed by H7N9 Hemagglutinin 1 (HA1) boosting elicited human antibodies with high neutralizing activities against H7N9 viruses in vitro. As a small ungulate, Tc caprine offers the advantages of low cost and quick establishment of herds, therefore complementing the Tc cattle platform in responses to a range of medical needs and diagnostic applications where small volumes of human antibody products are needed.

Legumes that contain condensed tannins may have lower ruminal protein degradation than alfalfa. The present study investigated the effects of feeding birdsfoot trefoil (Lotus corniculatus L.) hay on lactational performance and N utilization and excretion. Eight multiparous Holstein cows in midlactation (150 ± 22.3 d-in-milk) were randomly assigned to 2 treatments [alfalfa hay-based total mixed ration (AHT) or birdsfoot trefoil hay-based total mixed ration (BHT)] in a crossover design with 2 experimental periods. Each experimental period lasted 17 d (14 d of adaptation and 3 d of sampling and total collection). Hays comprised approximately 50% of DM in experimental diets. There were no treatment effects on dry matter intake (DMI; 21.4 vs. 20.7 kg/d), milk yield (29.4 vs. 28.1 kg/d), milk fat concentration (3.20% vs. 3.21%), and milk protein concentration (3.20% vs. 3.16%) for AHT and BHT, respectively. In addition, dietary treatments did not affect milk yield/DMI or energy-corrected milk yield/DMI. In contrast, apparent crude protein digestion decreased in cows fed BHT compared with those fed AHT (60.7% vs. 69.1%). Concentration of milk urea-N decreased by feeding BHT compared with AHT (11.9 vs. 13.3 mg/100 mL), whereas total N excretion did not differ between AHT and BHT diets. However, cows fed BHT excreted more N in feces (194 vs. 168 g/d), whereas urinary N excretion was lower compared with cows fed AHT. The shift of N to feces resulted in a decrease in urinary N:fecal N ratio in cows fed BHT relative to those fed AHT. Overall results in the current study suggest that feeding birdsfoot trefoil in dairy diets shifts routes of N from urine to feces compared with feeding alfalfa hay, with little effect on lactational performance. Reduction in urinary N and any impact on environment may be attributed to functional effect of condensed tannins in birdsfoot trefoil hay.

The hypothesis of this study was that the leukocyte populations and expression levels of genes related to immune response, growth factors and apoptosis would be altered at the fetal-maternal interface in somatic cell nuclear transfer (SCNT)-generated sheep pregnancies. Placental and endometrial samples from sheep pregnancies established by SCNT and natural breeding (control) were collected at 45 days and at term. Expression of genes related to growth factors, apoptosis and immune response was examined using quantitative reverse transcription polymerase chain reaction. Endometrial leukocyte populations and major histocompatibility class I (MHC-I) protein expression were examined by immunohistochemistry. At term we observed altered expression of genes related to apoptosis, growth factors and immune response in placental and endometrial tissue of SCNT pregnancies. In Day-45 pregnancies there was less-pronounced abnormal expression and only genes related to apoptosis and growth factors were abnormal in the placenta. Endometrial gene expression profiles were similar to age-matched controls. Placental MHC-I protein expression was similar in SCNT and controls at 45 days but increased in the SCNT at term. The altered gene expression at the fetal-maternal interface likely contributes to the placental dysfunction and overgrowth observed in sheep SCNT pregnancies.

Large animal models of osteoarthritis are a necessary testing ground for FDA approval of human medicine applications. Sheep models have advantages over other available large animals, but development and progression of osteoarthritis in sheep is exceedingly slow, which handicaps progress in development of potential treatments. We combined oblique angle forced exercise to increase stress on the stifle, with surgical destabilization to hasten the development of osteoarthritis in ewes. Methods for early detection of clinical signs included radiography, urine, and serum biomarker assays and gait analysis and ex vivo we used microcomputed tomography and macroscopic joint analysis. Our model was able to produce clinically detectable signs of osteoarthritis in a relatively short period (14 weeks). Changes in bone were highly correlated between microcomputed tomography and radiographic analysis and changes in cartilage correlated well between urinary glycosaminoglycan levels and serum aggrecanase analyses. Exercise improved the negative effects of destabilization in bone but exacerbated the negative effects of destabilization in cartilage. These observations suggest that wemay need to consider treatments for bone and cartilage separately. These results represent an improved large animal model of osteoarthritis with rapid onset of disease and superior detection of bone and soft tissue changes.

Large animal models of progressive atrial fibrosis would provide an attractive platform to study relationship between structural and electrical remodeling in atrial fibrillation (AF). Here we established a new transgenic goat model of AF with cardiac specific overexpression of TGF‐β1 and investigated the changes in the cardiac structure and function leading to AF.

Reduced developmental competence after IVF has been reported using oocyte derived from small follicles in several species including cattle, sheep, and goats. No information is currently available about the effect of follicle size of the cytoplast donor on in vivo development after somatic cell nuclear transfer (SCNT) in goats. Oocytes collected from large (≥3 mm) and small follicles (<3 mm) were examined for maturation and in vivo developmental competence after SCNT. Significantly greater maturation rate was observed in oocytes derived from large follicles compared with that of small follicles (51.6% and 33.7%, P < 0.05). Greater percent of large follicle oocytes exhibited a low glucose-6-phosphate dehydrogenase activity at germinal vesicle stage compared with small follicle oocytes (54.9% and 38.7%, P < 0.05). Relative mRNA expression analysis of 48 genes associated with embryonic and fetal development revealed that three genes (MATER, IGF2R, and GRB10) had higher level of expression in metaphase II oocytes from large follicles compared with oocytes from small follicles. Nevertheless, no difference was observed in pregnancy rates (33.3% vs. 47.1%) and birth rates (22.2% vs. 16.7%) after SCNT between the large and small follicle groups). These results indicate that metaphase II cytoplasts from small and large follicles have similar developmental competence when used in goat SCNT.

The present retrospective study investigated pregnancy rates, the incidence of pregnancy loss and large offspring syndrome (LOS) and immune-related gene expression of sheep and goat somatic cell nuclear transfer (SCNT) pregnancies. We hypothesised that significantly higher pregnancy losses observed in sheep compared with goat SCNT pregnancies are due to the increased amounts of T-helper 1 cytokines and proinflammatory mediators at the maternal–fetal interface. Sheep and goat SCNT pregnancies were generated using the same procedure. Control pregnancies were established by natural breeding. Although SCNT pregnancy rates at 45 days were similar in both species, pregnancy losses between 45 and 60 days of gestation and the incidence of LOS were significantly greater in sheep than in goats. At term, the expression of proinflammatory genes in sheep SCNT placentas was increased, whereas that in goats was similar to that in control animals. Genes with altered expression in sheep SCNT placentas included cytotoxic T-lymphocyte-associated protein 4 (CTLA4), interleukin 2 receptor alpha (IL2RA), cluster of differentiation 28 (CD28), interferon gamma (IFNG), interleukin 6 (IL6), interleukin 10 (IL10), transforming growth factor beta 1 (TGFB1), tumor necrosis factor alpha (TNF-α), interleukin 1 alpha (IL1A) and chemokine (C-X-C motif) ligand 8 (CXCL8). Major histocompatibility complex-I protein expression was greater in sheep and goat SCNT placentas at term than in control pregnancies. An unfavourable immune environment is present at the maternal–fetal interface in sheep SCNT pregnancies.

History A 2-year-old Quarter Horse filly was retired from race training in the fall of 2012 in preparation for breeding in 2013. To hasten the onset of seasonal ovulatory activity, the filly was exposed to an artificial long-day photoperiod (16 h of light/d) beginning December 1, 2012. On January 25, 2013 (day 0), transrectal palpation and ultrasonography were performed to assess the status of the filly’s reproductive tract. Examination revealed the left ovary was small (length, 31 mm; width, 12 mm; height, 23 mm), firm, and devoid of follicular activity. In contrast, the right ovary was considerably larger (diameter, approx 70 mm), lacked a palpable ovulation fossa, and was characterized ultrasonographically by a large, thick-walled cavity containing echogenic fluid. The uterus and cervix were flaccid, which was consistent with a seasonally anovulatory state and suggested that the right ovary did not contain functional luteal tissue producing progesterone. A stallion was not available for assessment of the filly’s reproductive behavior, but she was considered to be behaviorally normal when allowed contact with other mares.

Studies on patients, large animal models and transgenic mouse models have shown a strong association of atrial fibrosis with atrial fibrillation (AF). However, it is unclear whether there is a causal relationship between atrial fibrosis and AF or whether these events appear as a result of independent pathological changes in the heart. We are testing the hypothesis that goats that overexpress TGF-β1 (transforming growth factor beta1) specifically in cardiac myocytes will develop atrial fibrosis that in turn will lead to AF. Many aspects of AF-related remodeling have been studied comprehensively in goat models. However, these AF models are typically mechanically induced (eg, the rapid atrial pacing model). This unique transgenic goat model has the potential to offer insights into the role of fibrosis in AF initiation and progression without the confounding effects of mechanical AF induction. Somatic cell nuclear transfer (SCNT or cloning) was used to produce TGF-β1 transgenic pregnancies. First, pcDNA3.1DV5-MHC-TGF-β1cys33ser vector was constructed by subcloning the MHC-TGF-β1 fragment from the plasmid pUC-BM20-MHC-TGF-β1 into the pcDNA3.1D V5 vector. The NeonTM transfection system was used to electroporate primary goat fetal fibroblasts. After two weeks of G418 selection, the resulting G418 resistant colonies were screened by PCR to confirm transgene integration into goat genomic DNA. PCR positive cells were used for SCNT. Cloned embryos (n=264) were cultured for 12-60 h in vitro and then transferred into synchronized recipient females (n=15). Confirmation of pregnancy was done by ultrasonography on day 30 post-transfer. At the time of this abstract submission, 40% (6/15) of recipients were confirmed to be pregnant as determined by the presence of a heartbeat. The range for the stage of gestation is between day-60 and day-120. The first delivery date is April 28, 2012. Several reports documented no pregnancy losses after 30 days of gestation in goats. Therefore, we expect that most if not all of these pregnancies will result in delivery of live offspring. To our knowledge, this will be the first transgenic goat model generated for cardiovascular research.