ARF Successes

Identification of the Gene for Grey Coat Color in Alpacas.

Anim Genet. 2019 Oct;50(5):493-500. doi: 10.1111/age.12814. Epub 2019 Jul 12.

A non-synonymous SNP in exon 3 of the KIT gene is responsible for the classic grey phenotype in alpacas (Vicugna pacos).

M Jones, C Sergeant, M Richardson, D Groth, S Brooks, K Munyard

Abstract

The alpaca classic grey phenotype is of particular interest to the industry. Until now, there were only indirect data suggesting that the KIT gene was involved in the classic grey phenotype. All exons of KIT in three black and three classic silvergrey alpacas were sequenced. Five non-synonymous SNPs were observed. There was only one SNP found that was present only in the silvergrey alpacas, and this was also the only SNP predicted to be damaging. This variant results in a change of a glycine (Gly) to an arginine (Arg) at amino acid position 126 (c.376G>A), occurring in the second Ig-like domain of the extracellular domain of KIT. Basic protein modelling predicted that this variant is likely destabilising. Therefore, an additional 488 alpacas were genotyped for this SNP using the tetra-primer amplification refractory mutation system PCR (Tetra-primer ARMS-PCR). All classic grey alpacas were observed to be heterozygous, and 99.3% of non-grey dark base colour alpacas were found to be homozygous for the wildtype allele in this position. These results confirm that the classic grey phenotype in alpacas is the result of a c.376G>A (p.Gly126Arg) SNP in exon 3 of KIT. These data also support the hypothesis that the grey phenotype is autosomal dominant and that the mutation is most likely homozygous lethal.

Chemical in Seminal Fluid that Induces Ovulation has been Identified as Nerve Growth Factor

For a male animal to pass on his genes, he must create sperm. However, the fluid which carries sperm — semen — contains much more than just sperm cells. In 1985, a group of Chinese researchers found that when camel seminal fluid was injected into female camels, they ovulated, even when no sexual activity had occurred. In 2005, Gregg Adams, a veterinarian at the University of Saskatchewan in Saskatoon, Canada successfully repeated the Chinese experiment in llamas. Following 7 more years of work Dr. Adams and his colleagues at the Universidad Austral de Chile in Valdivia, Chile, have recently identified the chemical as a protein called — nerve growth factor, or NGF, which had been known to function in the brain to keep neurons alive. NGF from semen appears to send signals to the female llama brain that result in ovulation. The study was published in the Proceedings of the National Academy of Sciences August 20, 2012. "The idea that a substance in mammalian semen has a direct effect on the female brain is a new one," said Adams in a press release. "This latest finding broadens our understanding of the mechanisms that regulate ovulation and raises some intriguing questions about fertility." To learn more about the role of NGF in ovulation you can read the full article in the 2013 Herdsire edition of Alpacas Magazine or on the ARF website. Just click on Fertility in the Library. Dr. Adam’s studies were funded in part by the Alpaca Research Foundation.

An Integrated Radiation Hybrid Map of the Alpaca

Warren E. Johnson, PhD, Laboratory of Genomic Diversity
National Cancer Institute, NIH, Frederick, Maryland

Description:

Genetic maps are useful in studying which genes are linked to inherited traits and related topics such as infectious diseases, reproduction physiology, behavior, nutrition and evolutionary history. Genetic maps for humans, dogs and mice have increased the knowledge of these species; however, few genetic resources have been developed for camelids. This study is developing a map of the alpaca genome, which will help investigators identify inherited traits in these animals. A better understanding of these traits will immediately provide a benefit to the health of individual animals and entire herd management. A genetic map also opens the doors for future studies that will increase the knowledge of camelids.

Results:

Researchers developed several molecular genetic tools that will be used to further alpaca research. These included development of a radiation hybrid panel for mapping of genetic markers, which provides a preliminary map for analyzing relationships between alpaca, human and cow genomes, and development of probes for comparative cytogenetic analyses. These tools led to the alpaca being chosen by the National Cancer Institute for whole genome sequencing, a process that will be completed in 2007. This has led to the commitment of additional resources for alpaca research and has increased the number of researchers embarking on camelid research projects worldwide Future identification of genes and mutations will be critically important to alpaca breeders and researchers.

Read the investigator profile on Dr. Warren Johnson.

Analysis of the genome sequence of an alpaca corona virus

Jin L, Cebra CK, Baker RJ, Mattson DE, Cohen SA, Alvarado DE, Rohrmann GF., Virology, 365, 198-203, 2007. Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA. ling.jin@oregonstate.edu

Coronaviral infection of New World camelids was first identified in 1998 in llamas and alpacas with severe diarrhea. In order to understand this infection, one of the coronavirus isolates was sequenced and analyzed. It has a genome of 31,076 nt including the poly A tail at the 3' end. This virus designated as ACoV-00-1381 (ACoV) encodes all 10 open reading frames (ORFs characteristic of Group 2 bovine coronavirus (BCoV). Phylogenetic analysis showed that the ACoV genome is clustered closely (>99.5% identity) with two BCoV strains, ENT and LUN, and was also closely related to other BCoV strains (Mebus, Quebec, DB2), a human corona virus (strain 043) (>96%), and porcine hemagglutinating encephalomyelitis virus (>93% identity). A total of 145 point mutations and one nucleotide deletion were found relative to the BCoV ENT. Most of the ORFs were highly conserved; however, the predicted spike protein (S) has 9 and 12 amino acid differences from BCoV LUN and ENT, respectively, and shows a higher relative number of changes than the other proteins. Phylogenetic analysis suggests that ACoV shares the same ancestor as BCoV ENT and LUN.

Prevalence of bovine viral diarrhea virus (BVDV) infected US alpaca herds and factors associated with BVDV seropositive herd status

CL Topliff, EM Becker, DR Smith, SL Clowser, DJ Steffen, JN Henningson, BW Brodersen, D Bedenice, RJ Callan, C Reggiazdo, CL Kelling, VBMS, University of Nebraska Lincoln, Lincoln, NE

The objectives of this study were to determine the prevalence of BVDV infected US alpaca herds and factors associated with occurrence of BVDV seropositive herd status. Crias from 63 herds respresenting 26 states were tested for neutralizing antibodies, BVDV and BVDV RNA. Seventeen of the 63 herds (27%) had crias with BVDV neutralizing antibody. Four herds (6.3%) were identified as having persistently infected PI crias. Twenty herds (31.7%) reported recent serious disease problems. Factors significantly associated with BVDV seropositive herd status were feeding bovine colostrum and abortions. Another factor implicated from case studies contributing to a BVDV seropositive herd status was ingestion of colostrum from dams previously exposed to BVDV in other herds. These findings confirm the importance of BVDV infections in US alpacas and underscore the merit of adhering to sound herd biosecurity practices to avoid exposure to BVDV infected animals. In addition our study revealed that frequently a seropositive herd status is not attributable to the presence of infected animals within the herd and that other factors must be considered to determine the current BVDV infection status of a herd. This study has been accepted for publication in the Journal of the American Veterinary Association.

Read the investigator profile on Dr. Clayton Kelling.

Pharmacokinetics of Oral Omeprazole in Llamas

Poulsen KP, Smith GW, Davis JL, Papich MG. J. Vet Pharmacol Ther, 6, 539-543, 2005. Department of Population Health & Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA

Gastrogard, an oral formulation of omeprazole, was given to six llamas at a dose of 4 mg/kg once a day for 6 days. Plasma samples were collected at 0, 15, 30, 45, and 60 min and 2, 3, 4, 6, 8, 12, and 24 h on days 1 and 6. Plasma omeprazole concentrations were measured by high-pressure liquid chromatography with ultraviolet detection. Pharmacokinetic parameters calculated included the area under the curve (AUC(0-infinity)), peak plasma concentration (Cmax), time of peak plasma concentration (Tmax), and terminal half-life (t(1/2)). On day 6, plasma omeprazole concentrations reached a Cmax of 0.12 microg/mL at a Tmax of 45 min. The t(1/2) of omeprazole was 2.3 h and the AUC(0-infinity) was 0.38 h x microg/mL. Plasma concentrations remained above the minimum concentration for inhibition of gastric acid secretion projected from other studies on day 6 in all the llamas for approximately 6 h. However, the AUC(0-infinity) was below the concentrations associated with clinical efficacy. It was not possible to measure oral systemic bioavailability because there was no i.v. data collected from these animals. However, using data published on the i.v. pharmacokinetics of omeprazole in llamas, oral absorption was estimated to be only 2.95%. Due to low absorption the oral dose was increased to 8 and 12 mg/kg and studies were repeated. There were no significant differences in Cmax, Tmax, or AUC(0-infinity) for either of the increased doses. These results indicate that after 6 days of treatment with doses up to 12 mg/kg, oral omeprazole produced plasma drug concentrations which are not likely to be associated with clinical efficacy in camelids.

Read the investigator profile on Dr. Geoff Smith.

The Effect of Oral Omeprazole on Third Compartment pH in Clinically Normal Alpacas

Dr. Jennifer Lynn Johnson, University of Minnesota

Third compartment ulcers are a serious medical condition in stressed and sick camelids. Therefore, it is critical to the health of the camelid for ulcers to be treated quickly and effectively. The easiest way to treat ulcers is through the use of orally administered anti-ulcer medications. Previous work has shown that many of the anti-ulcer medications used in veterinary medicine, such as ranitidine and cimetidine, are ineffective in altering gastric pH in camelids. Moreover, in the previous study funded by ARF omeprazole was shown not to reach therapeutic levels in the blood stream when administered orally. Consistent with the work of Poulsen, et al, the results of the present study showed that oral omeprazole did not alter third compartment pH in alpacas.

Read the investigator profile on Dr. Jennifer Lynn Johnson.

Efficacy and Pharmacokinetics of Pantoprazole in Alpacas

Geof Smith, DVM, PhD; North Carolina State University College of Veterinary Medicine

In this study, 6 adult male alpacas were anesthetized and fitted with a third compartment cannula for measuring gastric pH. Following recovery, alpacas received 1 mg/kg pantoprazole intravenous every 24 hrs for 3 days or 2 mg/kg subcutaneously every 24 hrs for 3 days. All alpacas received both IV and SQ pantoprazole, with a minimum of 3 weeks between treatments. Third compartment pH was recorded at regular intervals and plasma samples were taken for pharmacokinetic analysis. Pantoprazole induced a slow but sustained increase in third compartment pH when given by both the IV and SQ routes. Baseline third compartment pH (1.81 + 0.7) increased to 2.47 + 0.8, 3.53 + 1, and 4.03 + 1.3 at 24, 48 and 72 hrs following IV administration. Third compartment pH increased from 1.73 + 0.6, at baseline to 3.05 + 1.1, 4.01 + 1.4, and 3.61 + 1.6 at 24, 48 and 72 hrs following SQ administration. This study showed that pantoprazole represents a safe and effective drug for increasing third compartment pH in alpacas. It is likely an effective treatment for third compartment ulcers and might be useful for prophylactic administration in stressed camelids at high risk for developing ulcers.

Pharmacokinetics After Intravenous, Subcutaneous, and Oral Administration of Enrofloxacin to Alpacas

Gandolf AR. Papich MG. Bringardner AB. Atkinson MW. American Journal of Veterinary Research. 66, 767-71, 2005.

Abstract

OBJECTIVE:

To determine plasma concentrations of enrofloxacin and the active metabolite ciprofloxacin after p.o, s.c., and i.v. administration of enrofloxacin to alpacas. ANIMALS: 6 adult female alpacas. PROCEDURE: A crossover design was used for administration of 3 single-dose treatments of enrofloxacin to alpacas, which was followed by an observational 14-day multiple-dose regimen. Single-dose treatments consisted of i.v. and s.c. administration of injectable enrofloxacin (5 mg/kg) and p.o administration of enrofloxacin tablets (10 mg/kg) dissolved in grain to form a slurry. Plasma enrofloxacin concentrations were measured by use of high-performance liquid chromatography. The multiple-dose regimen consisted of feeding a mixture of crushed and moistened enrofloxacin tablets mixed with grain. Behavior, appetite, and fecal quality were monitored throughout the 14-day treatment regimen and for 71 additional days following treatment.

Results:

Mean half-life following i.v., s.c., and p.o. administration was 11.2, 8.7, and 16.1 hours, respectively. For s.c. and p.o administration, mean total systemic availability was 90.18% and 29.31%, respectively; mean maximum plasma concentration was 3.79 and 1.81 microg/mL, respectively; and area under the curve (AUC) was 50.05 and 33.97 (microg x h)/mL, respectively. The s.c. or p.o administration of a single dose of enrofloxacin yielded a ratio for AUC to minimum inhibitory concentration > 100 for many grampositive and gram-negative bacterial pathogens common to camelids. Conclusions and Clinical Relevance-The administration of enrofloxacin (5 mg/kg, s.c., or 10 mg/kg, p.o) may be appropriate for antimicrobial treatment of alpacas.

Read the investigator profile on Dr. Rae Gandolph.

Ovulation-inducing factor in alpaca semen

Gregg P. Adams, DVM, PhD.; Western College of Veterinary Medicine; University of Saskatchewan; Canada

Camelids have been categorized as induced ovulators and present dogma suggests that physical stimulation during copulation is primarily responsible for eliciting ovulation. Recent discoveries, however, challenge this dogma. The project focuses on the isolation and characterization of an ovulation-inducing factor (OIF) present in the seminal plasma of camelids. The factor was initially reported in Bactrian camels, but has not been documented in any other species.

Studies were conducted to document the existence of an OIF in the seminal plasma of alpacas and llamas. In Experiment 1, female alpacas were given alpaca seminal plasma or saline intramuscularly or by intrauterine infusion. Only alpacas that were given seminal plasma intramuscularly ovulated (93%). In Experiment 2, ovulation was detected in 90% llamas at a mean of 29 hours after seminal plasma treatment. Plasma progesterone concentrations were maximal 9 days after treatment and back to minimum at 12 days after treatment. In Experiment 3, females were given seminal plasma, GnRH (positive control), or saline (negative control), and ovulation was detected in 100%, 83% and 0% in the respective groups. Blood samples taken every 15 minutes for 8 hours after treatment revealed that seminal plasma caused circulating luteinizing hormone (LH) to become elevated within 1 hour and remain elevated for over 8 hours. Compared to the GnRH group, the corpus luteum (CL; progesterone-producing gland in the ovary necessary for maintenance of pregnancy) grew larger and plasma progesterone concentration was twice as high in the seminal plasma group. Results show, for the first time, that a potent ovulation-inducing factor is present in the semen of alpacas and llamas. Treatment-induced ovulation was associated with a surge in circulating concentrations of LH and enhancement of CL form and function.

The existence and nature of this factor has direct implications on fertility, infertility, breeding management, and commercial development of therapeutic drugs for alpacas. The evolutionary conservation of such a factor raises the possibility of its existence in other induced and spontaneously ovulating species; hence, the characterization of OIF in the seminal plasma of alpacas may have much broader implications. Further studies are being conducted to isolate and characterize the chemical in semen of alpacas and to determine if it is present in other species.

Publications:

Ratto MH, Huanca W, Singh J, Adams GP. Comparison of the effect of ovulation-inducing factor (OIF) in the seminal plasma of llamas, alpacas, and bulls. Theriogenology 66, 1102-6, 2006.

Ratto MH, Huanca W, Singh J, Adams GP.Local versus systemic effect of ovulation-inducing factor in the seminal plasma of alpaca, .Reprod Biol Endocrinol, 3, 29, 2005.

Adams GP, Ratto MH, Huanca W, Singh J , Ovulation-inducing factor in the seminal plasma of alpacas and llamas. Biology of Reproduction 73:452-457, 2005.

Ratto MH, Huanca W, Huanca T, Singh J, Adams GP, Ovulation-inducing factor in seminal plasma: species comparison and molecular weight determination. Proceedings of the annual meeting of the Society for the Study of Reproduction, Vancouver, BC August 2004, Abstract 181.

Adams GP, Ratto MH, Singh J, Ovulation-inducing factor in the seminal plasma of llamas. International Congress on Animal Reproduction, Porto Seguro Brazil August 2004, p 217.

Ratto MH, Huanca W, Singh J, Adams GP., Effect of OIF on ovulation rate and luteal development in llamas. 1st Annual Reproductive Science and Medicine Research Symposium. March 3, 2005, p 18. (Best basic science paper, 2nd Place)