Comparing ovarian expression of sperm acrosome associated 3 protein in young and adult queens.

The purpose of this research was to quantify sperm acrosome associated 3 protein expression in the ovaries of young (3.0 ± 0.9 months, n = 11) and adult (10.4 ± 2.8 months, n = 11) queens. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded feline ovarian sections. Ovaries were obtained following routine ovariohysterectomy of queens. Cellular expression of sperm acrosome associated 3 protein was measured in primordial, primary, secondary, and tertiary follicles using an image-analysis software's red, green, and blue stack and manual thresholding functions. The oocyte nucleus, ooplasm, granulosa cells, and theca cells were outlined using the freehand selection tool and mean grey value was recorded. Results from each cellular location were compared between age groups using a Student's t-test and between follicle stages using an analysis of variance. Compared to adult queens, younger queens had significantly greater sperm acrosome associated 3 protein expression in granulosa cells of primary, secondary, and tertiary follicles. Also, theca cells of secondary and tertiary follicles had significantly greater sperm acrosome associated 3 protein expression in younger queens compared to adult queens. The oocyte nucleus of primordial, primary, and secondary follicles had significantly greater sperm acrosome associated 3 protein expression in younger queens compared to adult queens. However, sperm acrosome associated 3 protein expression within the ooplasm did not differ significantly between age groups of any follicle type. More research is needed to determine what role sperm acrosome associated 3 protein may play in female fertility in animals as well as what mechanisms regulate ovarian sperm acrosome associated 3 protein expression over time.

Ovine endometrial estrogen receptor expression is altered following PG-600 administration.

BACKGROUND: Dysregulation of the estrogen receptor 1 (ESR1) expression during the establishment of pregnancy could contribute to reduce fertility reported in ewes treated with PG-600. OBJECTIVES: The objective of this study was to evaluate the effect of treatment with PG-600 on ESR1 expression in the ovine endometrium during early diestrus. METHODS: Polypay ewes (n = 24) that had exhibited an oestrous cycle of normal duration (16-18 days) were treated with intravaginal progesterone-releasing device (CIDR) for 9 days. Cloprostenol was administrated two days before CIDR withdrawal. On the day of CIDR withdrawal (day 0), ewes received a 5 mL intramuscular injection of PG-600 (400 IU equine chorionic gonadotropin and 200 IU human chorionic gonadotropin) or saline. Blood samples were collected before treatment and on the day of tissue collection to determine serum estradiol-17ß and progesterone concentrations using radioimmunoassay. On days 4 and 7, six ewes from each treatment group were anaesthetised and a laparotomy was performed to obtain intercaruncular endometrial samples. Endometrial samples were collected ipsilateral to the ovary bearing the highest number of corpora lutea. An estradiol exchange assay was used to determine ESR1 concentration. RESULTS: Estradiol concentrations did not differ by time or treatment, but progesterone concentrations were significantly higher in the PG-600-treated group on day 7 despite a similar number of corpora lutea. Endometrial ESR1 concentration was significantly reduced on day 7 in the PG-600 group. CONCLUSIONS: In conclusion, although progesterone was higher than controls on day 7, ESR1 did not differ from controls suggesting that treatment with PG600 is unlikely to impair reproductive potential.

Understanding the effects of sustained supraphysiologic concentrations of luteinizing hormone in gonadectomized dogs: What we know and what we still need to learn.

Removal of the gonads with surgical sterilization results in a loss of negative feedback to the hypothalamus and anterior pituitary. The sustained supraphysiologic luteinizing hormone (LH) concentrations in gonadectomized dogs can significantly alter organ function and even induce neoplastic changes. For example, gonad removal has a profound effect on thyroid function and is reported to be the most significant cause for the development of hypothyroidism in dogs. Thirty percent more gonadectomized dogs develop hypothyroidism compared with intact dogs. Within the canine thyroid, LH receptors are co-localized with thyroid stimulating hormone (TSH) receptors. Continuous LH receptor activation in gonadectomized dogs may interfere TSH receptor function by consuming second messengers involved in G-protein receptor cell signaling, preventing the action of TSH when it binds to its receptor in the thyroid, resulting in hypothyroidism. The incidence of anterior cruciate ligament ruptures is significantly increased following gonad removal independent of breed, sex, weight or body condition. Luteinizing hormone receptors are expressed in the cruciate ligament and continuous LH receptor activation may increase laxity in these ligaments, resulting in joint instability. Both male and female gonadectomized dogs are at a significantly increased risk for lymphoma and hemangiosarcoma. Luteinizing hormone receptors are also abundant in these tissues. Research in four canine hemangiosarcoma cell lines found that LH receptor activation induces cell proliferation. In addition, research in three canine T-cell lymphoma cell lines found that LH receptor activation induces cell proliferation, adhesion, and invasion as well as increases LH receptor expression. Research is needed to determine if LH reducing strategies using gonadotropin releasing hormone agonists will increase remission times in gonadectomized dogs with LH receptor-positive tumors. In conclusion, among the non-reproductive functions of gonads, suppression of LH secretion and resulting LH receptor overexpression appear necessary in maintaining endocrine, musculoskeletal, and anti-neoplastic health.

Canine splenic hemangiosarcoma cells express and activate luteinizing hormone receptors in vitro.

OBJECTIVE: To determine luteinizing hormone receptor (LHR) expression and response to LHR activation in isolated canine splenic hemangiosarcoma cell lines in vitro. SAMPLES: In vitro cultures of commercially available canine splenic hemangiosarcoma cell lines (EFS, GRACE-HSA, and DAL-4). PROCEDURES: The percentage of each cell line expressing LHR was determined by immunocytochemistry. Cells were then treated with increasing doses (7.5 ng/mL, 75 ng/mL) of recombinant canine luteinizing hormone (cLH) for 48 hours and evaluated using a cell proliferation assay. RESULTS: The percentage of cells expressing LHR was 17.2 ± 4.5%, 11.8 ± 3.1%, and 6.9 ± 2.5% in EFS, GRACE-HSA, and DAL-4, respectively. There was significant increase in cell count in the DAL-4 and EFS cell lines following a 48-hour incubation at the highest cLH concentration (P = .028 and P = .019, respectively). There was not a significant increase in cell count in the GRACE-HSA cell line at either cLH concentration. CLINICAL RELEVANCE: Activation of LHR results in cell proliferation in some canine splenic hemangiosarcoma cell lines. These results may explain why spayed and castrated dogs with high circulating LH concentrations may develop hemangiosarcoma more frequently than intact dogs.

Luteinizing Hormone Receptor Expression in Neoplastic Mast Cells Is Increased in Spayed and Neutered Dogs.

Luteinizing hormone receptors (LHRs) are expressed in canine lymphoma and hemangiosarcoma. We hypothesized that LHR would be expressed in canine mast cell tumors (MCTs) and that more neoplastic mast cells would express LHR in gonadectomized dogs compared with intact dogs. Eleven archived formalin-fixed paraffin-embedded cutaneous MCT tissue sections were processed using routine immunohistochemistry. For both the KIT protein and LHR, the percentage of positive cells for each staining pattern (I-III) was calculated. A Student's t test was used to compare the total percentage of positive cells expressing LHR and KIT in intact and gonadectomized dogs. A one-way analysis of variance was used to compare the percentage of cells within each staining pattern for LHR and KIT in intact and gonadectomized dogs. All MCT expressed LHR. MCT from gonadectomized dogs had a significantly higher percentage of LHR-positive mast cells (84.2 ± 8.7%) compared with MCTs from intact dogs (64.3 ± 4.2%). This is the first study to demonstrate the expression of LHR in canine MCTs and to report that LHR expression is increased in neoplastic mast cells from gonadectomized dogs compared with intact dogs. Future studies are planned to evaluate the functionality of the LHR in canine neoplastic mast cells.

The effects of sex and neuter status on trauma survival in dogs: A Veterinary Committee on Trauma registry study.

OBJECTIVE: To determine the effect of sex and neuter status on trauma survival in dogs. DESIGN: Multi-institutional prospective case series, September 2013 to March 2019, retrospectively analyzed. SETTING: Level I and II Veterinary Trauma Centers. ANIMALS: Consecutive sample of 2649 dogs in the American College of Veterinary Emergency and Critical Care Veterinary Committee on Trauma patient registry meeting inclusion criteria. For inclusion, dogs had to have complete data entries, be postpubertal (≥7 months age in females and ≥10 months age in males), and have sustained moderate to severe trauma (animal trauma triage [ATT] score ≥5/18). Dogs that were dead upon arrival, euthanized for financial or unknown reasons alone, or that were presented by a Good Samaritan but subsequently humanely euthanized were excluded. MEASUREMENTS AND MAIN RESULTS: Data collected included age, sex, neuter status (intact, neutered), trauma type (blunt, penetrating, both), outcome (survived to hospital discharge, died, euthanized), and reason for euthanasia (grave prognosis, financial reasons, or both). Of 2649 eligible dogs, 56% survived to hospital discharge (n = 1469). Neutered females had a significantly higher survival rate (58.3% vs 51.3%; P = 0.03) compared to intact females, and neutered males had a significantly higher survival rate (56.6% vs 50.7%; P = 0.04) compared to intact males. There was no significant difference in survival between intact females and intact males (P = 0.87) or between neutered females and neutered males (P = 0.46). Mean cumulative ATT score was higher in intact groups and was found to be a significant predictor of survival (P < 0.01). Based on logistic models, overall odds of survival were 20.7% greater in neutered dogs. CONCLUSIONS: Gonadectomy is associated with lower ATT scores and improved survival after moderate to severe trauma in both female and male dogs.

Restoration of Reproductive Hormone Concentrations in a Male Neutered Dog Improves Health: A Case Study.

This case study reports on the use of hormone therapy to treat a dog with a range of physical and behavioral signs that began after gonadectomy. A male mixed breed dog neutered at 7 months of age presented at 1 year with health issues impacting quality of life. Reduced mobility, limping, rapid weight gain, and fear of unfamiliar people were treated over the next 3 years with trials of pain medication, joint supplements, thyroxine, antidepressant, and significant diet restrictions. Frequent carprofen administration and daily joint supplements reduced limping, but mobility was still poor. Weight stabilized on a strict diet but fear and anxiety responses to strangers continued to worsen. Hormone restoration therapy was initiated when the dog was almost 4 years of age. Weekly subcutaneous administration of testosterone cypionate (0.5 mg/kg) significantly reduced pain and increased muscle mass, thereby improving mobility. However, supraphysiologic concentrations of luteinizing hormone were not reduced with testosterone therapy so a gonadotropin-releasing hormone agonist was implanted. After hormone restoration, appetite was reduced, and anxiety and fear behaviors became manageable. The testosterone and gonadotropin-releasing hormone agonist treatment was easily administered, had no known side effects, and the owners were pleased with the outcome.

Investigating a method for pharmacologic semen collection in alpacas.

While semen evaluation is standard practice prior to a sale or when infertility is suspected in other species, it is rarely done in camelids due to the difficulties involved in collecting a sample. The reproductive physiology of alpacas differs to that of other domestic animals and is still poorly understood. In the stallion, a technique was developed for semen collection that pharmacologically induces ejaculation without copulation (ex copula). This study investigates whether semen could be reliably collected by ex copula ejaculation in male alpacas. Eleven male Huacaya alpacas were used in this study, and six ex copula treatment protocols were evaluated: (1) saline (control); (2) xylazine only (0.1 mg/kg); (3) xylazine only (0.2 mg/kg); (4) imipramine only (1.0 mg/kg); (5) imipramine (1.0 mg/kg) followed 10 minutes later with xylazine (0.1 mg/kg); and (6) imipramine (2.0 mg/kg) followed 10 minutes later with xylazine (0.1 mg/kg). Each treatment protocol was repeated two to five times. Azoospermic samples obtained from ex copula ejaculation contained numerous epithelial cells but no sperm. A reliable treatment for pharmacologically inducing ejaculation in alpacas remains to be found.

Estrus Synchronization in the Sheep and Goat.

Estrus synchronization and manipulation are a tool that has been used by producers to provide uniform lamb and kid meat production and dairy sheep and goat milk production, to concentrate work and labor cost, and to plan for the lambing and kidding time. Breeders can also use estrus synchronization to stimulate ewes and does to exhibit estrus and ovulate outside of the breeding season, although both the ovulation rate and pregnancy rate may be decreased. To increase the ovulation rate outside of the breeding season, a variety of estrus synchronization methods have been used.

Use of Hysteroscopy for Diagnosing Causes of Infertility in Camelids.

Hysteroscopy in alpacas and llamas allows for the identification of abnormalities on the surface or within the endometrium that cannot be identified with other methods. Hysteroscopy also allows for site-directed endometrial cytology, culture, and biopsy to achieve a definitive diagnosis. Even when no cause for infertility can be found, previously infertile females tend to become pregnant and maintain their pregnancies to term following the hysteroscopic procedure. This therapeutic effect may be a response to pre-hysteroscopy estrogen treatment, dilation of the uterine horns during hysteroscopy, and/or posttreatment uterine lavages. Complications following hysteroscopy have not been reported in camelids.

Resuscitation Compression for Newborn Sheep.

Neonatal maladjustment syndrome is characterized by depressed consciousness, neurologic signs, and reduced or nonexistent suckle reflex. Resuscitation compression (squeezing) has been used in newborn foals and calves to reduce the behavioral symptoms of neonatal maladjustment syndrome. In this review, the authors describe how resuscitation compression can be used in newborn sheep. This technique can improve abnormal neonatal lamb behavior and encourage suckling in resistant lambs.

Intramammary antibiotics with complementary acupuncture decreases milk serum N-acetyl-beta-D-glucosaminidase concentrations in dairy cattle with subclinical mastitis.

The objective of this research is to determine whether intramammary antibiotics with complementary acupuncture can reduce bovine mammary inflammation due to subclinical mastitis. Lactating cows were selected based on milk with a somatic cell count (SCC) greater than 500,000 cells/ml. Pre- and post-treatment milk samples were collected to determine SCC, aerobic bacterial content, milk ion conductivity, total protein, lactate dehydrogenase (LDH) and N-acetyl-beta-D-glucosaminidase (NAGase) concentrations. Milk serum was prepared from milk samples by double centrifugation. Concentrations of LDH and NAGase were determined using commercial enzyme-linked immunosorbent assays. Cows being treated with intramammary antibiotics were separated by random assignment to the acupuncture group (n = 10) and a no-acupuncture (control) group (n = 9). Both the acupuncture and control group were restrained for 30 min in a head catch 12 hr apart for a total of four times. For front quarters affected by subclinical mastitis, the acupuncture points used were spleen (SP) 12, SP 17, SP 18, SP 21, stomach (ST) 18 and conception vessel (CV) 12. For rear quarters affected by subclinical mastitis, the acupuncture points used were bladder (BL) 30, BL 30-1, BL 49, kidney (KI) 10, conception vessel (CV) 2 and CV 3. All parameters were compared using a Student t test. Significance was defined as p < .05. Compared to control cows, complementary acupuncture treatment reduced NAGase enzymatic activity in quarters of cows with subclinical mastitis. The reduction in NAGase suggests that complementary acupuncture treatment may be associated with healing of the damaged mammary epithelial cells, which are the primary source of NAGase activity in milk serum.

Developing a modified Apgar scoring system for newborn lambs.

The Apgar (appearance, pulse, grimace, attitude, respiration) score is an accepted method for assessing newborn viability in humans and domestic animals. The purpose of this research was to modify an existing Apgar scoring system for lambs and compare Apgar score with litter size and birth order, as well as time-to-stand and time-to-suckle. Polypay lambs (n = 75) delivered naturally were used for this study. Pulse (<100 bpm = 0; 100-175 bpm = 1; >175 bpm = 2) and respiration (<45% SpO2 = 0; 45-65% SpO2 = 1; >65% SpO2 = 2) were measured with a pulse oximeter attached to the lamb's ear. Appearance, grimace, and attitude were assessed using mucous membrane color (cyanotic = 0; pale = 1; pink = 2); response to nose stimulation (no response = 0; moves head slightly = 1; shakes head = 2); and response to rump stimulation (no movement = 0; moves but no attempt to stand = 1; attempts to stand = 2), respectively. The Apgar score was measured within 5 min of birth (T0), 15 min (T15) and 60 min (T60) after birth. The time-to-stand and time-to-suckle were recorded and compared to Apgar scores. Apgar scores did not differ by litter size or birth order. There were no significant correlations between the Apgar scores and time-to-stand. There was a moderate positive correlation between attitude and time-to-suckle at T0 (R2 = 0.467), which may affect the ingestion of adequate colostrum in a timely manner. Producers may choose to consider using Apgar scoring so that assistance can be provided to lambs before serious complications arise.

Gonad-Sparing Surgical Sterilization in Dogs.

Elective sterilization of pet dogs is a common surgical procedure performed in veterinary practice. The main benefit of sterilization is population control and the reduction in euthanasia of unwanted dogs. The most common methods for sterilizing female and male dogs are ovariohysterectomy (spay; which removes both the ovaries and the uterus) and castration (neutering; which involves removing the testicles), respectively. However, any surgery that removes the gonads changes the animal in both positive and negative ways. There is mounting evidence supporting the long-term health complications associated with surgical sterilization with gonad removal. Gonads are not merely gamete-producing or ancillary sex/reproductive organs but rather they are necessary endocrine glands for normal metabolic, behavioral, musculoskeletal, and anti-neoplastic health. The purpose of this mini review is to describe two gonad sparing surgeries that this author has used to sterilize dogs. These surgeries can be performed on pediatric patients without interfering with pubertal maturation. Dog owners can make the decision when the dog is completely mature whether or not the gonads should be removed.

Assessment of luteinizing hormone receptor expression in structural support tissues of canine hip and femorotibial joints.

OBJECTIVE: To determine whether luteinizing hormone receptors (LHRs) are expressed in canine femoral head subchondral bone (FHSB), hip joint round ligament (RL), cranial cruciate ligament (CCL), and femorotibial joint synovium (FJS) specimens. SAMPLE: 1 specimen each of the FHSB, RL, CCL, and FJS obtained from the left hind limbs of 19 fresh canine cadavers. PROCEDURES: 1 section of each FHSB, RL, CCL, and FJS specimen was processed with rabbit polyclonal IgG anti-human LHR antibody, and 1 section was treated with negative control reagents. Percentage immunoexpression of LHRs in FHSB and FJS sections was analyzed by assessment of 100 bone marrow cells or synoviocytes in 3 adjacent hpf (400×). In each RL and CCL section, immunoexpression of LHRs in fibrocytes was semiquantitatively analyzed on the basis of the mean of the product of percentage staining score (from 0 [no staining] to 3 [> 50% of cells stained]) and staining intensity score (from 0 [no staining] to 2 [moderate to strong staining]) for 3 adjacent hpf. RESULTS: All tissues examined had variable LHR expression. Expression of LHRs in FHSB, CCL, or FJS specimens did not differ between sexes or between sexually intact and gonadectomized dogs. However, RL specimens from female dogs had significantly greater LHR expression scores, compared with findings for male dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that LHRs are expressed in structural support tissues of canine hip and femorotibial joints. Further research is required to determine the LHRs' function, mechanism of action, and potential contribution to the pathogenesis of hip dysplasia or CCL rupture in dogs.

Possible Relationship between Long-Term Adverse Health Effects of Gonad-Removing Surgical Sterilization and Luteinizing Hormone in Dogs.

Spaying and neutering dogs is commonly used to prevent the birth of unwanted animals and eliminate the risk of reproductive diseases. However, removal of the gonads prevents the feedback of estrogen and testosterone on the pituitary and hypothalamus. As a result, luteinizing hormone (LH) is continuously elevated at supraphysiologic concentrations. Although the main role of LH is for reproductive function (e.g., ovulation), there are LH receptors present in several normal tissues including the thyroid and adrenal glands, gastrointestinal tract, cranial cruciate ligament and round ligament, and lymphocytes. In addition, there are LH receptors present in several neoplastic tissues (e.g., lymphoma, hemangiosarcoma, mastocytoma, transitional cell carcinoma, and osteosarcoma). The role of LH receptors in non-reproductive normal and neoplastic tissues is not known but may stimulate nitric oxide release and induce cell division. The precise etiology of the increased incidence of several non-reproductive long-term health complications following spaying and neutering is not known but may be related to LH receptor activation in these non-reproductive target tissues. How these effects may be mediated is described in this review.

miRWoods: Enhanced precursor detection and stacked random forests for the sensitive detection of microRNAs.

MicroRNAs are conserved, endogenous small RNAs with critical post-transcriptional regulatory functions throughout eukaryota, including prominent roles in development and disease. Despite much effort, microRNA annotations still contain errors and are incomplete due especially to challenges related to identifying valid miRs that have small numbers of reads, to properly locating hairpin precursors and to balancing precision and recall. Here, we present miRWoods, which solves these challenges using a duplex-focused precursor detection method and stacked random forests with specialized layers to detect mature and precursor microRNAs, and has been tuned to optimize the harmonic mean of precision and recall. We trained and tuned our discovery pipeline on data sets from the well-annotated human genome, and evaluated its performance on data from mouse. Compared to existing approaches, miRWoods better identifies precursor spans, and can balance sensitivity and specificity for an overall greater prediction accuracy, recalling an average of 10% more annotated microRNAs, and correctly predicts substantially more microRNAs with only one read. We apply this method to the under-annotated genomes of Felis catus (domestic cat) and Bos taurus (cow). We identified hundreds of novel microRNAs in small RNA sequencing data sets from muscle and skin from cat, from 10 tissues from cow and also from human and mouse cells. Our novel predictions include a microRNA in an intron of tyrosine kinase 2 (TYK2) that is present in both cat and cow, as well as a family of mirtrons with two instances in the human genome. Our predictions support a more expanded miR-2284 family in the bovine genome, a larger mir-548 family in the human genome, and a larger let-7 family in the feline genome.