Víctor H. Parraguez

Prediction of gestational age by ultrasonic fetometry in llamas (Lama glama) and alpacas (Lama pacos)

Fetal biparietal diameter (BPD) and thorax height (TH) were measured by ultrasound during intrauterine growth in pregnant llamas (Lama glama) and alpacas (Lama pacos). The goal was to establish representative curves that allows estimation of gestational age (GA) from real-time ultrasonic measurements of these fetal structures at any stage of gestation. Llamas and alpacas were mated under controlled conditions. Ultrasound exams were conducted to determine pregnancy status 1 month later. Measurements of fetal BPD and TH were conducted from the second month of pregnancy until term. Observation and assessment of fetal TH was difficult during the last 3 months of pregnancy, specially in llamas. Regression curves were calculated from the data as a function of GA, with the best fit represented by the following equations: llama GA=(BPD-0.002399)43.02293,r=0.98,P<0.001; llama GA=(TH-0.07137)46.94485, r=0.95,P<0.001; alpaca GA=(BPD-0.11376)47.23287, r=0.98,P<0.001; alpaca GA=(TH-0.36436)52.87663, r=0.96,P<0.001, where GA was measured in days and BPD and TH in centimeters. Results indicate that ultrasonic measurement of these fetal biometric variables constitute a valuable tool to estimate GA at any stage of pregnancy in these domestic South American camelids.

Perinatal neuroendocrine regulation. Development of the circadian time-keeping system

During gestation, the perinatal neuroendocrine axis keeps clock time. In primates, the suprachiasmatic nucleus (biological clock in mammals), shows oscillatory function by midgestation. There is evidence in rodents that the mother, during pregnancy, entrains the fetal suprachiasmatic nucleus (SCN) and newborn circadian rhythms. We are investigating the role of maternal melatonin as an entraining signal for the newborn circadian time-keeping system in the Cebus apella (New World non-human primate). Twenty-four hour rhythms of temperature and cortisol are present in the 4 days old C. apella newborn. Preliminary data suggests that inhibition of maternal melatonin by exposing pregnant females to constant light alters these rhythms. We have found binding sites for melatonin and expression of mRNA for Mel 1A receptor in hypothalamus, kidney and testis. These preliminary results suggest that maternal melatonin may play a role in relating the perinatal circadian time-keeping system to environmental signals.

Mitochondrial distribution and meiotic progression in canine oocytes during in vivo and in vitro maturation

The objective was to evaluate mitochondrial distribution, and its relationship to meiotic development, in canine oocytes during in vitro maturation (IVM) at 48, 72, and 96 h, compared to those that were non-matured or in vivo matured (ovulated). The distribution of active mitochondria during canine oocyte maturation (both in vitro and in vivo) was assessed with fluorescence and confocal microscopy using MitoTracker Red (MT-Red), whereas chromatin configuration was concurrently evaluated with fluorescence microscopy and DAPI staining. During IVM, oocytes exhibited changes in mitochondrial organization, ranging from a fine uniform distribution (pattern A), to increasing clustering spread throughout the cytoplasm (pattern B), and to a more perinuclear and cortical distribution (pattern C). Pattern A was mainly observed in germinal vesicle (GV) oocytes (96.4%), primarily in the non-matured group (P < 0.05). Pattern B was seen in all ovulated oocytes which were fully in second metaphase (MII), whereas in IVM oocytes, ∼64% were pattern B, irrespective of duration of culture or stage of nuclear development (P > 0.05). Pattern C was detected in a minor percentage (P < 0.05) of oocytes (mainly those in first metaphase, MI) cultured for 72 or 96 h. In vitro matured oocytes had a minor percentage of pattern B (P < 0.05) and smaller mitochondrial clusters in IVM oocytes than ovulated oocytes, reaching only 4, 11, and 17% of MII at 48, 72, and 96 h, respectively. Thus, although IVM canine oocytes rearranged mitochondria, which could be related to nuclear maturation, they did not consistently proceed to MII, perhaps due to incomplete IVM, confirming that oocytes matured in vitro were less likely to be competent than those matured in vivo.

Early pregnancy diagnosis in alpaca (Lama pacos) and llama (Lama glama) by ultrasound

An ultrasonography study of early pregnancy diagnosis was carried out in 19 alpacas and 12 llamas, after controlled matings. The aim was to determine the earliest gestational age at which pregnancy diagnosis by transrectal ultrasonography could be achieved, and to generate an empirical formula for gestational sac diameter (GSD) growth as a function of gestational age (GA), allowing an estimate of GA during the first month of pregnancy. We found that pregnancy diagnosis may be carried out as early as 9 days after mating in alpacas and 7 days in llamas. This diagnosis was found to be accurate at 23 days in alpacas and 34 days in llamas. The empirical relations that best describe the relationship between GSD and GA were GA = logGSD + 1.2339/0.0585 r = 0.85; P < 0.001 in alpacas, and GA = logGSD + 1.2649/0.0546 r = 0.77, P < 0.001 in llamas, where GA is measured in days and GSD in centimeters. Our results also indicate that ultrasonography is a reliable technique for early pregnancy diagnosis. Furthermore, the empirical formulae reliably make it possible to estimate GA from GSD during the first month of pregnancy and their use might improve the efficiency of camelid breeders.

Effects of antioxidant vitamins on newborn and placental traits in gestations at high altitude: comparative study in high and low altitude native sheep

The present study evaluated the hypothesis that the effects of hypoxia on sheep pregnancies at high altitude (HA) are mediated by oxidative stress and that antioxidant vitamins may prevent these effects. Both HA native and newcomer ewes were maintained at an altitude of 3,589 m during mating and pregnancy. Control low altitude (LA) native ewes were maintained at sea level. Half of each group received daily oral supplements of vitamins C (500 mg) and E (350 IU) during mating and gestation. Near term, maternal plasma vitamin levels and oxidative stress biomarkers were measured. At delivery, lambs were weighed and measured, and placentas were recovered for macroscopic and microscopic evaluation. Vitamin concentrations in supplemented ewes were two- or threefold greater than in non-supplemented ewes. Plasma carbonyls and malondialdehyde in non-supplemented ewes were consistent with a state of oxidative stress, which was prevented by vitamin supplementation. Vitamin supplementation increased lamb birthweight and cotyledon number in both HA native and newcomer ewes, although placental weight and cotyledon surface were diminished. Placentas from vitamin-supplemented HA ewes were similar to those from ewes at sea level, making these placental traits (weight, number and diameter of cotyledons) similar to those from ewes at sea level. Vitamin supplementation had no effect on LA pregnancies. In conclusion, supplementation with vitamins C and E during pregnancy at HA prevents oxidative stress, improving pregnancy outcomes.

VEGF overexpression in the astroglial cells of rat brainstem following ozone exposure.

Ozone, a major photochemical pollutant, produces rapid damages in the pulmonary airway tract and in the central nervous system. This study focused on the neural mechanisms underlying the adaptive responses to an acute ozone exposure. Vascular endothelial growth factor (VEGF) is a factor associated with cellular recovery following brain injury. The aim of this study was to assess and localize the cellular expression of VEGF, since the central respiratory areas show a neuroplasticity in response to ozone. Adult rats were subjected to 0.5ppm ozone for 3h and then recovered for further 3h. The expression of VEGF was evaluated by immunocytochemistry in the central respiratory areas, i.e., the nucleus tractus solitarius (NTS) and the ventrolateral medulla (VLM). The data show a VEGF overexpression at the end of ozone exposure, which persisted during the 3-h recovery. Interestingly, using confocal analysis the bulk of VEGF labeling was observed in astroglial cell bodies and branches, while neuronal labeling was hardly noticed. Moreover, VEGF colocalized with IL-6 and TNFalpha in astrocytes closely apposed to blood vessel walls. The vasculature area was markedly increased (+58%) during post-ozone recovery. The data show that an acute ozone exposure affects primarily glial cells in the central nervous system. The VEGF up-regulation which persists after ozone exposure may contribute to brain repair and consecutive functional adaptations.

Fertility in a high-altitude environment is compromised by luteal dysfunction: the relative roles of hypoxia and oxidative stress

BackgroundAt high altitudes, hypoxia, oxidative stress or both compromise sheep fertility. In the present work, we tested the relative effect of short- or long-term exposure to high altitude hypobaric hypoxia and oxidative stress on corpora luteal structure and function.MethodsThe growth dynamics of the corpora lutea during the estrous cycle were studied daily by ultrasonography in cycling sheep that were either native or naïve to high-altitude conditions and that were supplemented or not supplemented with antioxidant vitamins. Arterial and venous blood samples were simultaneously drawn for determination of gases and oxidative stress biomarkers and progesterone measurement. On day five after ovulation in the next cycle, the ovaries were removed for immunodetection of luteal HIF-1alpha and VEGF and IGF-I and to detect IGF-II gene expression.ResultsThe results showed that both short- and long-term exposure to high-altitude conditions decreased luteal growth and IGF-I and IGF-II gene expression but increased HIF-1 alpha and VEGF immunoexpression. The level of plasma progesterone was also increased at a high altitude, although an association with increased corpus luteum vascularization was only found in sheep native to a high-altitude location. Administration of antioxidant vitamins resulted in a limited effect, which was restricted to decreased expression of oxidative stress biomarkers and luteal HIF-1alpha and VEGF immunoexpression.ConclusionsExposure of the sheep to high-altitude hypobaric hypoxia for short or long time periods affects the development and function of the corpus luteum. Moreover, the observed association of oxidative stress with hypoxia and the absence of any significant effect of antioxidant vitamins on most anatomical and functional corpus luteum traits suggests that the effects of high altitude on this ovarian structure are mainly mediated by hypoxia. Thus, these findings may help explain the decrease in sheep fertility at a high altitude.

Steroidogenesis in sheep pregnancy with intrauterine growth retardation by high-altitude hypoxia: effects of maternal altitudinal status and antioxidant treatment.

Sheep pregnancy in high-altitude environments frequently involves hypoxia and oxidative stress and causes intrauterine growth retardation. The adverse effects of altitude on fetal growth can be prevented by the administration of antioxidant vitamins, but the mechanisms responsible are not well known. The maintenance of a viable pregnancy depends largely on adequate placental steroidogenesis, especially in the last two-thirds of pregnancy. Thus, in the present study we evaluated the effect of antioxidant vitamins (C and E) on plasma concentrations of progesterone and 17β-oestradiol during the last two-thirds of high-altitude pregnancies in ewes both native and naïve to the high-altitude environment. In addition, pregnancy outcomes were evaluated by determining the bodyweight of newborn lambs. Sex steroid patterns differed between ewes with and without vitamin supplementation. Concentrations of plasma progesterone and 17β-oestradiol were significantly higher in the supplemented groups from approximately 40 days before parturition until near term. Newborn weights were significantly lower in animals not adapted to the higher altitude, and vitamin supplementation prevented this decrease. In conclusion, the administration of antioxidant vitamins in the present study enhanced placental steroidogenesis, thus favouring fetal development in pregnancies developing at high altitudes.

In vitro fertilization and development of cumulus oocytes complexes collected by ultrasound-guided follicle aspiration in superstimulated llamas.

The objective was to evaluate the developmental competence of cumulus-oocyte complexes (COC) collected by follicular aspiration in llamas treated with FSH or eCG. Llamas were assigned randomly to two groups (n = 16 per group) and treated, at the time of ovarian follicular wave emergence, with either: 1) 25 mg of FSH im, twice daily for 4 d; or 2) 1000 IU of eCG as a single i.m. dose. The start of gonadotropin treatment was considered Day 0. Both groups were given 5 mg of Armour Standard LH im on Day 6, and COC were collected by follicle aspiration on Day 7. Expanded COC collected from FSH- (n = 157) and eCG-treated llamas (n = 151) were fertilized in vitro using epididymal sperm, and presumptive zygotes were in vitro cultured in SOF medium for 8 d. The FSH and eCG treatment groups did not differ with respect to: the number of follicles ≥7 mm (16.0 ± 2.7 vs 14.0 ± 1.9, respectively; P = 0.5); the number of COC collected (11.5 ± 1.9 vs 9.7 ± 1.2; P = 0.4); the number of expanded COC (9.8 ± 1.4 vs 9.4 ± 1.2; P = 0.8); or the percentage of presumptive zygotes which developed into 2 to 8 cell stage embryos (65.3 vs 63.1), morulas (46.2 vs 42.5), or blastocysts (23.1 vs 20.5; P > 0.05). In conclusion, FSH and eCG treatments were equally effective for recovery of a high number of expanded COC which were used directly for in vitro fertilization. Furthermore, rate of embryo development was not significantly affected by the gonadotropin treatment used.

Plasma progesterone in alpaca (Lama pacos) during pregnancy, parturition and early postpartum

Plasma progesterone concentration during pregnancy and the early postpartum period was measured by radioimmunoassay in five alpacas (Lama pacos), of the Huacaya breed, whose pregnancy length had a mean of 344.8 +/- 4.4 days. Concentration of progesterone increased (P < 0.001) from low premating values (0.11 +/- 0.06 nmol/l) to greater values at 30 days of pregnancy (8.05 +/- 1.13 nmol/l) and remained high up to 2 months of pregnancy. A slight transitory decline was observed between 3 to 7 months. Concentration of plasma progesterone dropped markedly during the 72 h before parturition especially, at the day of parturition.