Selective control of the estrous cycle of the dog through suppression of estrus and reduction of the length of anestrus.

The objective of this study was to determine the effectiveness of testosterone in suppressing estrus in the bitch, and of cabergoline in shortening the length of the subsequent anestrous period. In Experiment 1, 12 diestrual Beagle bitches were randomly divided into two groups when plasma progesterone (P(4)) concentration was <1 ng/ml (Day 0). Starting on Day 0, bitches in Group 1 (n=6) were treated with testosterone cypionate every 14 days for a total of 239 days, and bitches in Group 2 served as untreated controls. On Day 274, bitches in both groups were treated with cabergoline for 40 days and blood samples were obtained on Days 274, 276 and 279 for determination of plasma prolactin (PRL) concentrations using RIA. All bitches were observed for proestrual bleeding during treatment with cabergoline. In Experiment 2, 12 Greyhound bitches previously treated with testosterone within the last 6 months were randomly divided into two groups. At the initiation of this experiment, P(4) concentration was determined to verify that all bitches had a concentration of <1 ng/ml (Day 0). Starting on Day 0, bitches in Group 1 (n=6) were treated with cabergoline for 36 days, and bitches in Group 2 (n=6) served as untreated controls. Blood samples were obtained on Days 0, 2 and 5 to determine PRL concentrations. All bitches were observed for proestrual bleeding during treatment with cabergoline. In Experiment 1, one bitch (Group 1) exhibited estrus after treatment with testosterone (1mg/kg body weight) for 43 days, and one bitch (Group 1) exhibited estrus after treatment with testosterone (2mg/kg body weight) for 113 days. None of the other four bitches in Group 1 exhibited estrus during the period of testosterone treatment (239 days). All bitches in Group 2 (control) exhibited estrus during the 239 days of the study. In addition, five of the six testosterone-treated bitches showed signs of proestrual bleeding within an average of 12.6 days (range of 5-25 days) after treatment with cabergoline; and, four of the six nontestosterone bitches showed signs of proestrual bleeding within an average of 28 days (range of 6-46 days). Prolactin concentrations in bitches in both Groups 1 and 2 significantly decreased after treatment with cabergoline. In Experiment 2, one of the six bitches showed signs of proestrual bleeding within 15 days after treatment with cabergoline. From the results of this study, it was concluded that exogenous testosterone was moderately effective (66%) in suppressing estrus in Beagle bitches, and cabergoline was effective in shortening the length of the anestrous period of Beagle bitches whose estrous cycle was previously suppressed with exogenous testosterone, but less effective in shortening the length of the anestrous period in Greyhound bitches previously treated with testosterone to suppress estrus.

Effects of prepubertal gonadectomy on physical and behavioral development in cats.

OBJECTIVE: To determine the effect of prepubertal gonadectomy on physical and behavioral development in cats. DESIGN: Prospective controlled study of kittens randomly assigned to 3 treatment groups: group 1, neutered at 7 weeks of age; group 2, neutered at 7 months of age; and group 3, sexually intact controls. ANIMALS: 31 clinically normal male and female kittens. PROCEDURE: Age at distal radial physeal closure and mature radius length were determined radiographically. Six behavioral characteristics were recorded monthly. At 1 year of age, body weight was recorded and thickness of the falciform ligament was measured from a lateral abdominal radiographic view. Secondary sex characteristics were also examined at 1 year of age. RESULTS: There were no differences between group-1 and group-2 cats for any of the study variables. Sexually intact cats (group 3) weighted significantly less than group-2 cats and had less falciform fat and earlier distal radial physeal closure than cats of both neutered groups. Group-3 cats manifested greater intraspecies aggression, less affection, and greater development of secondary sex characteristics than neutered cats. CLINICAL IMPLICATIONS: Neutering cats at 7 weeks of age had similar effects on physical and behavioral development, compared with neutering at the more traditional age of 7 months. These data lend support to the concept of prepubertal gonadectomy, already performed by many animal shelters/humane organizations, as a method of enhancing the effectiveness of pet population control programs.

Purification and characterization of a uterine retinol-binding protein in the bitch.

A major canine endometrial secreted protein (cP6, 23,000-M(r)) was purified by ion-exchange and gel-filtration chromatography and characterized by two-dimensional gel electrophoresis. Anti-[human retinol-binding protein (hRBP)] serum identified cP6 on immunoblot analysis and immunoprecipitated cP6 from culture medium. This major protein was also shown to bind [3H]retinol. N-terminal and internal amino acid sequences were determined and compared with previously identified protein, RNA, or DNA sequences. N-terminal analysis revealed that cP6 had high identity and similarity to serum retinol-binding proteins (RBPs), while internal sequence analysis showed a strong similarity to rat androgen-dependent epididymal protein and beta-lactoglobulins. Amino acid analysis, however, showed significant differences between these proteins and cP6 in both total amino acid content and certain selected amino acids. Immunohistochemical analysis showed staining for RBP only in the uterine luminal epithelium. These studies suggest that bitch endometrium secretes a family of proteins (cP6), some of which bind [3H]retinol, are immunologically related to the RBP family, and have N-terminal and internal sequences with a high similarity to RBP, beta-lactoglobulins and other members of the lipocalin family. This family of proteins may be important in early development for supplying retinol or derivatives to the developing embryo.

Identification and immunolocalization of proteins synthesized by dog endometrium and membranes.

Previous studies on cyclic and pregnant bitches on dioestrus days 3-10 indicated synthesis de novo of at least ten protein complexes, two of which are major protein groups (cP5, M(r) 54,600; cP6, M(r) 23,000). Protein expression differed by day but not by status, suggesting that the embryo does not affect uterine protein synthesis. Ovariectomized bitches treated with various steroid regimens showed induction of cP5 and cP6 only after oestrogen priming followed by progesterone. Recently, N-terminal amino acid microsequencing has identified cP6 as a member of the retinol-binding protein (RBP) family. The present study was designed (a) to identify and characterize proteins synthesized de novo in explant culture from cyclic (dioestrus days 10-16) and early pregnant (dioestrus days 10-26) dogs, (b) to examine distribution of proteins by endometrium preimplantation (PI), and between (BI) and at (I) implantation sites; (c) to characterize proteins synthesized by embryonal membranes and (d) to localize RBP and CUPED (a cat oestrogen-dependent uterine glycoprotein) immunocytochemically in the uterus and embryonal membranes. Proteins synthesized by endometrium were examined by incorporation of [3H]leucine or [35S]methionine on two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by fluorography or autoradiography. Nine proteins, characterized by electrophoresis and previously found between dioestrus days 3-10, appeared to be expressed through day 16 of cyclic and day 26 of pregnant dioestrus, although many decreased and cP5 was lost after day 13. In BI and I sites, RBP decreased in number of isoelectric variants as gestation progressed, while the major M(r) form (23,000) was reduced to 21,500. There was no change in M(r) and isoelectric variants of RBP from cyclic dogs, suggesting differences in RBP gene regulation. Proteins synthesized by embryonal membranes appeared to be serum proteins. Immunolocalization of RBP confirmed that oestrogen priming followed by progesterone was required for induction. In pregnant bitches, staining was present in all luminal epithelia on dioestrus day 10, but by day 17 only specific epithelium stained. As pregnancy progressed to day 24 of dioestrus, staining was localized to specific epithelial cells of the deep spongy zone and yolk sac. In ovariectomized bitches, CUPED was confirmed to be oestrogen dependent with staining in both luminal and glandular epithelia.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis of uterine endometrial proteins during early diestrus in the cyclic and pregnant dog, and after estrogen and progesterone treatment.

The objectives of this study were to identify and characterize dog uterine endometrial proteins synthesized de novo in explant culture during early luteal phase, to examine distribution of these proteins prior to the embryo's entering the uterus and during its free-floating period prior to implantation, and to examine regulation of endometrial proteins by estrogen and progesterone (P4) treatments. Uterine endometrium was collected from cyclic and pregnant bitches on diestrus Days 3, 7, and 10 as determined by loss of cornification of vaginal epithelium, and from ovariectomized dogs after treatment with corn oil, estrogen, P4, or estrogen followed by 1 or 2 wk of P4. Tissue was incubated in an explant culture system in the presence of [3H]leucine or [35S]methionine. The rate of incorporation of [3H]leucine into nondialyzable macromolecules indicated no significant change in rates of incorporation by status (pregnant vs. nonpregnant), day, or steroid treatment. Uterine endometrial-conditioned culture medium, analyzed by two-dimensional SDS-PAGE and fluorography, revealed a complex array of at least ten proteins or protein complexes in cyclic and pregnant bitches. No difference in protein pattern was detected by status; however, differences in distribution were apparent by day of cycle or early pregnancy. Two major proteins, cP5 (M(r) 54,686) and cP6 (M(r) 23,010) appeared to be differentially expressed. Expression of cP5, maximal on diestrus Day 3, decreased as the cycle or pregnancy progressed to diestrus Day 10. In contrast, expression of cP6, a minor protein on diestrus Day 3, appeared to be up-regulated for each status to Day 10, with increased intensity and multiple isoelectric and molecular-weight variants. In ovariectomized steroid-treated dogs, two-dimensional SDS-PAGE showed that pattern and distribution of specific proteins were affected by treatment. Acidic protein cP1 (M(r) 87,600), synthesized after corn oil and P4 treatment, was suppressed with estradiol (E2). Proteins cP2 (M(r) 40,000 and M(r) 42,000), present with all treatments, were intensified with P4. A high-M(r) basic protein complex (cP3) and acidic protein cP4 were expressed with E2 and maintained with P4 treatment. Proteins cP5 and cP6, while not induced by E2 or P4 alone, required E2 priming for P4 induction. Protein cP5 was down-regulated while cP6 was up-regulated with P4 for 2 wk. Proteins induced by estrogen followed by 1 or 2 wk of P4 treatments were similar to those released by endometrial explants collected from pregnant and cyclic bitches on Days 3, 7, and 10 of spontaneous diestrus.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of feline conceptus proteins during pregnancy.

This study characterized proteins secreted de novo by feline conceptuses collected on Days 10, 12, and 15 (n = 22, preimplantation blastocysts); Days 15, 16, 17, 19, 21, and 25 (n = 6, postimplantation zonary girdle [ZG] i.e. trophoblast and endometrium); and Days 30, 36, 39, and 50 (n = 5, postimplantation ZG and free chorioallantois [CA]) and cultured in Minimal Essential Medium. De novo secretion was shown by incorporation of 3H-leucine into proteins detected in culture media by 2D-PAGE and fluorography. Western blotting, and NH2-terminal amino acid microsequencing. Major radiolabeled proteins identified as they appeared temporally on fluorographs were as follows: feline conceptus protein 1 (fCP1), Mr = 20,000, pI 5.0-5.3; fCP2, Mr = 80,000, pI 6.5-7.2; fCP3a, Mr = 67,000, pI 6.3-6.5; fCP3b, Mr = 67,000, pI 5.9-6.3; fCP4, Mr = 56,000, pI 5.0-6.0; and fCP5, Mr = 29,000, pI 5.0-5.8. The fCP1 was produced by blastocysts on Days 10-15, ZG on Days 16-25, and CA on Day 30; on Days 39-50, CA synthesized 5 proteins, possibly fCP1 isomers. The fCP2, fCP3a and b, and fCP4 were produced by blastocysts on Day 15, ZG on Day 25, and CA on Days 30-50. The fCP5 was made by ZG on Days 16-36 and by CA on Days 30-39. Western blotting identified fCP1 as retinol-binding protein (RBP), fCP2 as alpha fetoprotein, fCP3a as albumin, and fCP3b as transferrin. Amino acid sequence homologies between fCP1 and rabbit and human plasma RBP and porcine conceptus RBP2 were 93, 96, and 100%, respectively, at the first 37 NH2-terminal amino acids. The identities of fCP4 and fCP5 have not been established. Antiviral activity detected in all media was less than 3 units/ml when tested with feline fibroblast cells infected with vesicular stomatitis virus.

Metabolites of estradiol in serum, bile, intestine and feces of the domestic cat (Felis catus ).

We wished to develop an efficient, noninvasive method for monitoring ovarian function in domestic and nondomestic Felidae. We hypothesized that the method could be based on measurement of one of the major excreted estrogen metabolites. To identify and characterize the major excreted metabolites, a bolus of (14)C-estradiol was administered into the femoral vein of adult female cats. We measured the amounts of total radioactivity per unit time contained in unconjugated and conjugated estradiol metabolites, in conjugated metabolites that were hydrolyzable, and in those not hydrolyzable by beta Glucuronidase / aryl sulfatase (the enzyme). Radionuclide levels were determined in voided feces and urine, in jugular vein plasma, bile, contents of the duodenum, and in the small intestine. Metabolites of (14)C-estradiol were voided preferentially in feces and in equal amounts either as unconjugated estradiol or as conjugates not hydrolyzable by the enzyme. In plasma, conjugated estrogens comprised an increasing proportion of the total radioactivity during the first 40 min after administration. Plasma pools of samples from 0.5 to 30 min and 40 to 360 min contained a monoconjugate and a diconjugate, respectively; both were hydrolyzable by the enzyme. Bile and intestinal samples were collected at 360 min after administration. In the bile, 99% of the total radioactivity was in conjugated compounds, only 20% of which were not hydrolysable by the enzyme. The proportion of unconjugated metabolites increased to 18% in the duodenum and to 45% in the small intestine. The major conjugates contained in voided feces not hydrolyzable by the enzyme were estradiol sulfate (m/z = 351.6836), distributed as the 3-sulfate (20%) and 17-sulfate (80%); of the latter, 70% were 17alpha- and 30% 17beta-estradiol sulfates. These data document the fate of estradiol in the circulation of the cat, they demonstrate that a large portion of the voided estradiol metabolites are not hydrolyzable by the enzyme, and account for those conjugates previously termed nonhydrolyzable.

Induction of estrus in greyhound bitches with prolonged idiopathic anestrus or with suppression of estrus after testosterone administration.

Twelve anestrous adult Greyhound bitches were used to study a regimen for induction of estrus. Once daily, 7 bitches were given diethylstilbestrol (DES; 5 mg, PO) until sanguineous vaginal discharge and vulvar edema were observed (designated as day 1 of proestrus) and for 2 days thereafter. If no response was elicited after 7 days, a doubled DES dose was given for up to an additional 7 days. Luteinizing hormone (5 mg, IM) was given on day 5 of proestrus, and follicle-stimulating hormone (10 mg, IM) was given on days 9 and 11 of proestrus. Bitches were bred once on day 13. Five bitches were used as a control group; they were given candy tablets for 7 days (first day on tablets, treatment day 1) and 0.9% NaCl (1.0 ml, IM) on treatment days 12, 16, and 18. The 7 bitches treated with DES had a mean proestrus period of 7.7 days and a mean estrus period of 5.7 days up to the day of mating. After mating, they had a mean gestation interval of 64 days and delivered a mean of 4 pups/litter. In 5 bitches, initial treatment with 5 mg of DES/day induced proestrus within 7 days; however, in 2 bitches, additional treatment with 10 mg of DES/day was needed for 5 and 6 days, respectively. Serum estradiol-17 beta and progesterone concentrations remained at base line during the period of DES treatment. Concentrations of both hormones increased after injection with luteinizing hormone and remained high for the next 4 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Fetal survival in the cow after pregnancy diagnosis by palpation per rectum.

It is desirable to determine whether a cow has failed to become pregnant as early as possible, preferably prior to 50 d after insemination. Although palpation per rectum has been the generally accepted method of pregnancy diagnosis in cattle, the procedure may be a significant iatrogenic cause of fetal attrition. In a study conducted at a North Florida dairy from January through June 1982, pregnancy was determined in 192 Holstein-Friesian cows by measuring low milk progesterone (P4) content on day of insemination (Day 0) and elevated P4 on Days 21 and 24. Pregnant cows were randomly assigned to a treatment and a control group. Cows in the treatment group (n=85) were palpated per rectum twice between Days 42 and 46 after insemination. Cows in the control group (n=107) were not palpated until both groups were palpated at Day 90. Palpation, done by two experienced clinicians, consisted of palpation of fetal fluid fluctuation, identification of the amniotic vesicle, and slipping of the chorioallantoic membranes. In both groups of cows fetal viability was monitored by milk P4 content. Last milk (5 to 15 ml) was collected from one front quarter on Days 0, 21, and 24 and twice weekly thereafter through Day 63. Milk was defatted by centrifugation and the fat-free milk progesterone content measured by a radioimmunoassay without extraction. The milk P4 test was 80.0% accurate in determining pregnancy in the palpated and nonpalpated cows. In the cows palpated on Days 42 to 46, pregnancy rates declined by 7.5% as determined by palpation at Day 90, or by 11.4% as determined by milk P4 content through Day 63 (both values P<0.05). Cows that were not palpated on Days 42 to 46 showed a 1.9% increase or a 4.3% decline in pregnancy rates as determined by the same criteria. Before palpation, at Days 42 to 46, pregnancy rates were better in cows that were inseminated in winter (January to March) than in spring (April to June) (82.3% vs 61.6%; P<0.05); P4 content was higher (winter>spring=2.13 ng/ml vs 1.38 ng/ml; P<0.05). First-lactation cows had higher P4 values on Days 21 and 24 than older cows (P<0.01).

The use of ultrasonography for pregnancy diagnosis in the bitch.

Twenty-three Greyhound bitches housed at 3 breeding kennels were examined for pregnancy via transabdominal palpation and ultrasonography. Pregnancy was timed from the calculated day of ovulation (day of ovulation = day when first pup was whelped--63), and from a single breeding date (day 0). Starting on day 10 after ovulation, 9 bitches were monitored every 3 days by ultrasonography only, to determine gestational vesicle sizes during gestation and the time when fetal movements and heartbeats could be first detected. The other 14 bitches were examined by ultrasonography and transabdominal palpation on the same day every week, starting on postovulation day 19, to compare the effectiveness of the 2 methods of pregnancy determination. Parturition was the final determinant of pregnancy status. The earliest correct diagnosis of pregnancy was at 18 days after ovulation, but fetal movements and heartbeat could not be identified until days 28 and 35, respectively. Estimation of fetal numbers by ultrasound or palpation was not reliable when there were more than four in the litter. Pregnancy and nonpregnancy were correctly determined by both methods in an increasingly greater number of bitches as gestation progressed, but ultrasonography was more accurate at all stages; on days 19 to 22, 26 to 30, 34 to 38, and greater than 40 after ovulation, correct diagnoses were made in 33%, 42%, 50%, and 75% of the bitches by palpation and in 42%, 67%, 75%, and 83% of the bitches by ultrasonography. The most common error was failure to detect pregnancy by palpation or ultrasonography in bitches with small litters and tense abdominal muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Amikacin sulfate in the cat: serum, urine and uterine tissue concentrations.

Puerperal metritis and pyometra in non-breeding cats is frequently caused by gram-negative bacteria that are resistant to a variety of antibiotics. Amikacin has been found to be effective against pathogens associated with uterine infections in the mare and the woman, but its efficacy has not been studied in the cat. Serum concentrations of amikacin were determined in healthy adult cats (six male and six female) after administration of 5, 10, and 20 mg/kg body weight of amikacin sulfate, each dose given subcutaneously (s.c.), intramuscularly (i.m.) and intravenously (i.v.) to each of the cats using a repeat treatment design. In a subsequent experiment, the six females were given 10 mg/kg s.c. amikacin and samples of blood, urine and full-thickness uterine wall were taken at 40 and 120 minutes after treatment. Mean serum concentrations of amikacin peaked between 30 and 45 minutes after i.m. injection and between 45 and 60 minutes after s.c. injections. The serum amikacin concentration curves were similar regardless of dose or administration route except for a slightly longer retention time after the 20 mg/kg dose given i.m. and s.c. After s.c. injection of 10 mg/kg, the mean uterine concentration of amikacin at two hours after treatment was 4.1 ug/g; the concurrent mean serum concentration was 18.6 ug/ml.

Efforts to induce estrus in the bitch, using pituitary gonadotropins.

Induction of fertile estrus in 14 anestrous bitches was attempted, using pituitary gonadotropins given by intramuscular injection in 3 treatment regimens: as a single dose of 10-mg follicle stimulating hormone (FSH; 5 bitches), as multiple doses of FSH (1, 2, 4, 8, 16 mg) given to 4 bitches on days 1 and 2, 3 and 4, 5 and 6, 7 and 8, and 9 and 10, and as multiple doses of combined FSH and luteinizing hormone (LH; 1:1, 1:1, 2:1, 4:1, 8:2.5, 16:5 mg) given to 5 bitches on days 1, 3, 5, 7, 9, and 11, respectively. Four bitches were injected with equivalent volume of isotonic NaCl solution (saline). Treatments were evaluated by observation of sexual behavior in presence of a male, microscopic changes in exfoliated vaginal epithelium, plasma concentrations of estradiol-17 beta and progesterone, and qualitative and quantitative, gross and microscopic changes in the ovaries and uterus. Four of the 14 treated bitches came into estrus and mated (one became pregnant), two after single, and two after multiple doses of FSH. In each of the four, vaginal cornification, plasma estradiol and progesterone patterns, and ovarian and uterine changes were comparable with those seen in the one saline-treated bitch with a spontaneous estrous cycle and pregnancy. Three treated bitches manifested proestrous behavior (2 after a single injection and 1 after multiple injections of FSH) but had no measurable response in the vaginal epithelium, endocrine patterns, or appearance of ovaries and uterus. Seven gonadotropin-treated bitches and 3 saline-treated bitches did not respond in any way.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of abortion in the bitch with a synthetic prostaglandin analog.

Eight mixed-breed bitches in the middle (30 to 35 days) and late 3rd (53 to 56 days) of gestation were given 20 microg of synthetic prostaglandin analog/kg of body weight, IM (4 bitches) or the polyethylene glycol vehicle, IM (4 bitches). Bitches treated with prostaglandin aborted 2 days after treatment, whereas bitches given the vehicle delivered live pups at term. Luteolysis occurred within 24 hours of treatment, as shown by rapidly declining plasma progesterone concentrations. Microscopic signs of luteal degeneration were seen in ovaries removed 5 days after treatment. Three bitches erroneously designated as pregnant were treated during anestrus and had no marked changes in plasma progesterone or in gross and microscopic appearance of the reproductive tract. Serum alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase activities were increased slightly in 4 bitches on the day after treatment, but values declined to base-line values 24 hours later. Treatment side effects included panting, salivation, vomiting, repeated defecation, and transient pain at the injection site.

Ovarian and endocrine responses in the cat after coitus.

LH release leading to ovulation was induced in 17 of 29 oestrous periods. The time of ovulation after coitus was determined by histological examination or by observation at laparotomy of ovaries in situ. Histological methods revealed that ovulation was complete in most follicles (9 of 13) at 32 h post coitum and in all follicles that were involved in the ovulatory process by 36 h. When laparotomy was used, no signs of preovulatory change were noted at the first observation time, 22 h post coitum, but in 4 cycles in which the entire process of ovulation was observed, the ovulatory process occurred between 23 and 28 h (3 follicles), 23 and 27 h (2 follicles), 25 and 28 h (3 follicles), and 25 and 29 h (3 follicles) post coitum. The first ovulatory process noted was complete at 25 h post coitum. In cats, LH release continued over a 16-h period before returning to baseline (long surge), values being 616 +/- 180 ng/ml at 1/2 h and 941 +/- 154 ng/ml at 2 h post coitum. In 6 cats the LH release pattern was limited to a 4-h period (short surge), values being 537 +/- 218 ng/ml at 1/2 h and 353 +/- 245 ng/ml plasma at 2 h and basal (49 +/- 18 ng/ml) by 4 h post coitum. Decreased secretion of oestrogen by follicles in animals undergoing ovulation was first observed at 16 h post coitum. It is concluded that coitus induces LH release within minutes in the cat and that ovulation begins about 24 h later and finishes by about 32 h post coitum. Only one coital input can cause LH release for as long as 16-20 h although shorter periods of LH release (4 h or less) can result in ovulation.