Expression of the μ Opioid Receptor and Effects of the Opioid Antagonist Naloxone on In Vitro Maturation of Oocytes Recovered from Anoestrous Bitches

The μ-opioid receptor (MOR) is expressed in bovine, human, equine and canine oocytes, and in seasonal breeders, it is expressed with higher intensity during the anoestrous phase. Supplementation of in vitro maturation (IVM) medium with opioid agents, agonists or antagonists, was shown to affect oocyte maturation in several species such as rat, bovine and equine. This study reports the effects of supplementing IVM medium with naloxone (Nx), an opioid antagonist, on nuclear and cytoplasmic maturation rate of oocytes recovered from anoestrous bitches. Cytoplasmic maturation was examined in terms of mitochondrial (mt) distribution. In order to confirm the receptor-mediated action of Nx, in oocytes of anoestrous bitches, MOR expression was analyzed by Western blot. Cumulus–oocyte complexes, recovered from the ovaries of bitches in anoestrous, were cultured in vitro and Nx was added at the concentrations of 1 × 10⁻⁶, 1 × 10⁻⁸ and 1 × 10⁻¹⁰  m . The rate of oocytes resuming meiosis after culture in presence of 1 × 10⁻⁶  m Nx (29%) was significantly higher than that of oocytes of control group (12%; p < 0.05). However, treatment with Nx did not affect mt distribution pattern. In denuded oocytes and in corresponding cumulus cells, a doublet of 65 and 50 kDa was observed. We conclude that, in oocytes of anoestrous bitches, MOR is expressed and Nx significantly improves nuclear maturation rate. Further studies should be performed to elucidate the expression of other opioid receptors, such as δ and κ, and possible interactive effects of their antagonists on canine oocyte maturation.     

Second Oestrus Synchronization and Precocious Embryo Viability after Puberty Induction in Gilts by the Use of Gonadotrophin Treatment

The use of exogenous gonadotrophins in puberty inducement and ovulation synchronization is a technique that has a positive influence on the management of swine. The purpose of this study was to verify the effects of a second gonadotrophin treatment [equine chorionic gonadotrophin (eCG) and luteinizing hormone (LH), intramuscularly (i.m.)] upon the second oestrus synchronization and fertility in gilts. Seventy-one NAIMA (Pen Ar Lan) gilts had their first oestrus (puberty inducement) induced by a hormonal treatment (eCG and LH). Then, they were randomly distributed into two treatments, with (T1) and without (C) gonadotrophin treatment at the second oestrus. The animals were fed with a single ration (16% of crude protein and 3286.73 kcal ME/kg), and timed artificial insemination performed at the second oestrus. Gilts were slaughtered for embryo recovery and ovary examination about 5 days after insemination. There was no evidence of a difference in the percentage of the second oestrus expression (T1 - 90.90% and C - 86.84%), the duration of the oestrus cycle (T1 - 19.62 +/- 0.82 days and C - 19.67 +/- 4.14 days), the percentage of follicular cysts (T1 - 15.15% and C - 18.42%) and number of ovulations (T1 - 14.60 +/- 5.7 and C - 13.23 +/- 4.8) between treatments (p > 0.05). However, the hormonal treatment (T1) showed minor oestrus dispersion and embryo viability (T1 - 8.4 +/- 5.6 and C - 11.2 +/- 4.6) (p < 0.05). These results indicate that the better synchronization and expression of the second oestrus when using gonadotrophins (eCG and LH) is followed by a lower embryo viability, which is probably the consequence of the heterogeneous follicle recruitment during the injection of eCG.     

3D Reconstruction of the Mouse's Mesonephros

The present work reports on the three-dimensional reconstruction of the segmented mesonephros during the embryonic development of the mouse. With a light microscope and an automatic reconstruction of surfaces, aspects of the mesonephros are described. These surfaces are obtained by using digitized contour lines. A new interpolation method called DSI (Discrete Smooth Interpolation) enables correction of the distortion induced by microtomy in paraffin sections. After a tri-angulation step, this method uses a smoothing algorithm, which implies a spatial redistribution of the vertices of the triangles to correct the rotational and translational misalignment. The use of this 3D program improves the understanding of the development patterns and helps us to appreciate changes in the rebuilt mesonephros. By 10.5 embryonic days, tubules emerge from the Wolffian body and begin their formation, then between 11.5 and 13.5 embryonic days, tortuous mesonephric tubules bound to the Wolffian duct form small curls, which grow and finally unwind. At the same time, mesonephric tubules unbound to the Wolffian duct appear, and on 13.5 embryonic days, the Müllerian duct is visible. After 14.5 embryonic days, the segmented mesonephros keeps its general aspect but decreases in size. At this time, each gonad is provided with both Wolffian and Müllerian ducts. Later, the Wolffian duct differentiates into the definitive male duct system, whilst the Müllerian duct regresses. Conversely, the paramesonephric duct differentiates into the definitive female duct system, whilst the mesonephric duct in turn degenerates. By this time degeneration has begun in the cranial portion of the mesonephros and this process progresses caudally. The spatial organization of the mesonephric tubules and the precise organization of all connections between these elements and the ducts may be well defined. Such approach can allow for a high definition of the normal pattern of mesonephros differentiation.     

Non‐indigenous fish in protected spaces: Trends in species distribution mediated by illegal stocking

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Invasive tilapia juveniles are associated with degraded river habitats

• 1. Invasions are a main concern for conservationists. Identification of causes that raise or promote an invader's success is one of the first steps for preventing and/or fighting against invasions.
• 2. In the Brazilian River Paraiba do Sul, juveniles of invasive tilapia Oreochromis niloticus were detected associated with degraded habitats (mainly pollution from various sources). The relative abundance of juveniles of the competitor native species pearl cichlid Geophagus brasiliensis, morphologically and ecologically very similar, was negatively associated with habitat degradation.
• 3. A species‐specific molecular marker was employed to permit unambiguous identification of these small juveniles (2–3 cm long), which represent the most vulnerable life stage of both species.
• 4. It is suggested that adaptation to degraded habitats is one of the characteristics of invasive species, and consequently habitat conservation and restoration may help to prevent fish invasions.

Copyright © 2008 John Wiley & Sons, Ltd.     

Incidence of Unilateral Fertilizations after Low Dose Deep Intrauterine Insemination in Spontaneously Ovulating Sows under Field Conditions

A new procedure for non-surgical deep intrauterine insemination (DUI) in unrestrained sows hormonally induced to ovulate, has been reported. In comparison with standard artificial insemination (AI), with this procedure, the sperm numbers inseminated can be reduced 20-fold without reducing the reproductive performance of these hormonally treated sows. The present study evaluated, using two experiments, the reproductive performance applying 20-fold different sperm numbers per AI dose using DUI or standard AI in spontaneously ovulating sows, under field conditions. In experiment 1, AI was applied to crossbred sows at 12, 24 and 36 h after onset of spontaneous oestrus using one of the following two regimes: (i) DUI (treatment) with 0.15 x 10(9) fresh boar spermatozoa in 5 ml of Beltsville thawing solution (BTS) extender (n = 95), and (ii) standard cervical AI (control) with 2.85 x 10(9) fresh spermatozoa in 95 ml of BTS extender (n = 95). The farrowing rates of the two groups of sows were statistically similar (NS). However, a decrease (p < 0.002) in litter size and the total number of pigs born alive was observed in sows inseminated with the DUI procedure. In experiment 2, 42 post-weaned oestrus sows were inseminated following the same design described for experiment 1 during spontaneous oestrus. On day 6 after onset of oestrus, the proximal segment of the uterine horns of the sows were flushed under surgery to retrieve eventual embryos and evaluate the success of fertilization per cornua (e.g. occurrence of effective uni- vs bilateral sperm transport rendering uni- or bilateral, complete or partial fertilization). Retrieved embryos were assessed for cleavage and number of accessory spermatozoa. Although identical overall pregnancy rates were achieved in both insemination groups, the percentage of sows with partial bilateral fertilization and unilateral fertilization was markedly higher (p < 0.05) in the DUI group (35%) compared with the control (standard AI) group (5%), with a consequent lower (p < 0.001) percentage of viable early embryos after DUI. The number of accessory spermatozoa in the zona pellucida of the embryos was highly variable, but higher (p < 0.001) in control animals than in DUI-AI. No accessory spermatozoa were found in oocytes retrieved from sows depicting unilateral fertilization. In conclusion, DUI in spontaneously ovulating sows with 0.15 x 10(9) spermatozoa renders similar farrowing rates but a lower litter size compared with use of standard AI with a 20-fold higher sperm dose. The lower litter size ought to be related to a decreased distribution of spermatozoa after DUI leading to a higher incidence of partial bilateral and unilateral fertilization.     

Milk Progesterone Profile at and after Artificial Insemination in Repeat-Breeding Cows: Effects on Conception Rate and Embryonic Death

The aim of this study was to investigate whether the skim milk progesterone concentrations at artificial insemination (AI) and day of rise of post-ovulatory progesterone concentration thereafter affect the conception and embryonic death rates in repeat-breeding cows. Milk samples were obtained from 96 repeat-breeding cows that failed to conceive to three or more AIs. The samples were taken from the cows at the day of AI and three times/week until day 45 post-AI. Skim milk was obtained after centrifugation and used for progesterone assay. The cows with a progesterone concentration more than 0.5 ng/ml at AI showed a significantly higher incidence of late embryonic death than those having a progesterone concentration<0.5 ng/ml at AI (p<0.01). As the progesterone level at insemination rose, conception rate declined. A negative correlation was shown between conception rate and skim milk progesterone level at AI. Of 56 cows showing a rise of progesterone to 1 ng/ml or more within 6 days after AI, 28 cows (50%) conceived. On the contrary, only eight of 39 cows (20.5%) conceived when the progesterone rose up to 1 ng/ml after day 6 post-AI. We concluded that increased progesterone concentration at the time of AI and delayed rise of progesterone post-AI might lead to decrease in fertility in repeat-breeding cows.     

Pathological Conditions of the Reproductive Organs of Male Stray Dogs in the Tropics: Prevalence, Risk Factors, Morphological Findings and Testosterone Concentrations

The objective of this study was to estimate the prevalence of and risk factors for pathological conditions of the reproductive organs in stray dogs under tropical conditions. Three hundred and eighteen dogs were examined post-mortem in the period from 1 July 2002 to 30 June 2003. Before killing, a blood sample (from the cephalic vein) for testosterone assay was taken. Pathological conditions of the reproductive organs were found in 135 of the dogs (42.5%) and in 175 of the testes (64.8%). The most frequent pathologies found were testicular degeneration, cryptorchidism, testicular hypoplasia and testicular tumours (in 15.1%, 6.6%, 6.6% and 5.4% of the dogs and 15.1, 4.6, 6.0 and 3.5 of the testes, respectively). Transmissible venereal tumour (TVT) was seen in 5.4% of the dogs. Testicular degeneration was more common in old dogs and underweight dogs (p < 0.05). Testicular tumours were 14.3 times more common in cryptorchid dogs. Age was another important factor for the development of testicular tumours (p < 0.05). Lower levels of testosterone concentration (p < 0.05) were observed in dogs with advanced testicular degeneration (0.7 +/- 0.8 nM), dogs with hypoplastic testicles (0.8 +/- 0.9 nM) and dogs with one degenerated and one retained testis or with bilateral cryptorchidism (1.2 +/- 0.9 nM) compared to dogs with one or two normal testes (7.0 +/- 5.5 nM). Testicular volume and weight were significantly lower in degenerated, hypoplastic and retained testes compared with the contralateral normal testis. Some spermatogenic activity was found in three of the retained testes, producing oligozoospermic smears with a high percentage of sperm abnormalities. No comparable epidemiological data about male pathological conditions of the reproductive organs in the dog is available. The prevalence found in this study, yet, appears high.     

Ultrastructural Changes in the Adrenal Cortex of Rats Exposed to Extremely Low Temperatures

The response of the adrenal cortex to low temperature in relation to the length of the exposures is studied. The morphological changes of the adrenal cells, mainly in the Zona fasciculata, are examined. Thirty white rats of both sexes, divided into six groups, were exposed during time intervals of different lengths to temperatures ranging between – 12° and – 15°C. The cellular modifications encountered by means of the electron microscope at the level of the mitochondria, the smooth endoplasmic reticulum and the liposomes are analyzed and compared with the functional situation of the gland in an effort to establish the existence of a correlation between the morphology and the function.     

聚丙烯酰胺防治坡地水土流失田间试验研究

坡地篱用聚丙烯酰胺可以减少地表径流,提高土壤含水量,促进土壤沉降,增强土壤结构性和水稳性,因而提高了土壤的持水性和抗蚀性,减少了土壤侵蚀量和肥力流失量,有利于作物增产。施用聚丙烯酰胺可使产量增加１８．７％～３２．４％。施用聚丙烯酰胺对环境无不良影响,而且投入费用较低,是一条值得探索的防治坡地水土流失的新途径。