Effect of Maternal Flavour Conditioning Combined with Organic and Inorganic Iron-Supplemented Creep Feed on Piglet Performance and Haemoglobin Status.

Iron injections are vital but imperfect against iron deficiency anaemia (IDA). This experiment explored the effects on piglets of maternal flavour conditioning and the voluntary intake of anise flavoured, iron-supplemented creep feed compared with iron injections. The experiment was a 2 × 2 factorial arrangement: ±maternal exposure to dietary anise flavour and ±intramuscular injections of piglets. Twenty-three sows and their litters (242 piglets) were randomly allocated to one of four treatments (n = 5 or 6 per treatment): no flavour plus no injection (NF + NI); no flavour plus iron injection (NF + I); flavour plus no injection (F + NI); and flavour plus iron injection (F + I). All piglets could access anise flavoured, iron-supplemented creep feed (organic and inorganic forms) from D2 of birth. Sow feed intake and milk anethole concentration, piglet body weight (BW) and average daily gain (ADG), creep feed disappearance, piglet behavioural time budgets, and piglet blood glucose and haemoglobin concentrations were determined. Over the four-week study, the only significant differences found were that iron-injected piglets had reduced blood glucose (p = 0.036) on D14 and that maternal flavour provision increased the frequency of piglet creep feed interaction (p = 0.023) and decreased the frequency of suckling events (p = 0.009). In summary, maternal flavour conditioning reduced piglet creep feed neophobia without influencing consumption. The supplementation of creep feed with iron and anise flavour to piglets under the conditions of this trial was effective in preventing IDA, regardless of exposure to maternal flavouring conditioning.

Vaginal and vestibular electrical resistance as an alternative marker for optimum timing of artificial insemination with liquid-stored and frozen-thawed spermatozoa in sows.

Development of a pen-side test to objectively determine the ideal time for artificial insemination (AI) in the sow would save producers time and money. Current processes rely on identification of oestrus via subjective behavioural and physiological markers that are indicative of high blood oestrogen concentrations. This study attempted to use measurements of electrical resistance (ER) in the cervical mucus to pinpoint timing of AI accurately enough to lead to equivalent pregnancy rates as a natural mating. Thirty-six sows were divided into 3 groups and observed for signs of oestrus. Seven sows did not show any oestrus behaviour and were excluded from the study. The remaining 29 sows were inseminated via natural mating and conventional oestrus detection (n = 14), or inseminated artificially with either liquid-stored semen (n = 8) or frozen-thawed semen (n = 7) according to timing indicated from electrical resistance measurements in the vagina and vestibule. Sows that were artificially inseminated on the basis of the electrical resistance readings had a lower pregnancy rate (P = 0.034) and less piglets born alive per litter (P < 0.05) than those that were naturally mated according to a conventional oestrus detection regime. However, the pregnancy rate and total piglets born alive did not differ between the two groups that underwent artificial insemination. Change in electrical resistance in the vagina has the potential to accurately predict ovulation timing, but more work is required to refine the timing of AI in relation to the readings before the technique can be adopted by industry.

Defining "Normal" in Pig Parturition.

Animal production industries rely on efficient and successful reproductive outcomes, with pigs being no exception. The process of parturition in pigs (farrowing) can be especially prolonged, due to the large numbers of piglets being born (on average, approximately 13 piglets per litter in Australian conditions). Difficulties in farrowing (dystocia) lead to poor piglet outcomes and health problems in sows, in turn, causing economic loss for producers and welfare concerns for the animals. Despite the importance of this topic and publications in the area stretching back nearly 50 years, there is still no consensus on the prevalence of dystocia in pigs nor on how to identify a pig experiencing the condition. Understanding the process of parturition and the factors that influence its success is a crucial step towards the early identification of sows undergoing dystocia and development of best practices to assist them. This article describes the key factors that contribute to successful farrowing and identifies areas in which more research is required before the parturition process in the pig can be fully understood.

Artificial insemination with fresh, liquid stored and frozen thawed semen in dromedary camels.

This study was conducted to evaluate various factors affecting fertility following insemination of dromedary camels. In experiment 1, camels were either bred by natural mating (NM) or inseminated in the body of uterus with whole, split (50:50) or 1 mL of undiluted ejaculate. In experiment 2, camels were inseminated with fresh diluted semen either in the body of the uterus or tip of the uterine horn and at either the time of ovulation induction (0 h), 24 or 30 h later. In experiment 3, camels were inseminated at the tip of the uterine horn with different doses of fresh diluted semen (75, 150 or 300 x 106 motile spermatozoa) or with 150 x 106 motile spermatozoa diluted with different extenders (Green buffer, Optixcell or Triladyl). In experiment 4, camels were inseminated in the tip of the uterine horn with diluted (Triladyl or Optixcell) liquid-stored semen or diluted (Triladyl) frozen-thawed semen consisting of either 300 or 500 x 106 motile spermatozoa. The pregnancy rate in camels bred by NM was similar to camels inseminated with whole undiluted ejaculates whereas insemination with 1 mL undiluted ejaculate resulted in lower pregnancy compared to whole and split undiluted ejaculates (P < 0.05). Deposition of semen in the uterine body resulted in lower pregnancy rates compared to deposition in the tip of the horn (35.3% versus 72.2%, P < 0.05) but insemination at the time of ovulation induction and 24 h later resulted in higher pregnancy rate to camels inseminated at 30 h after induction (68.4 and 70.0% versus 23.5%; P < 0.05). Artificial insemination with 75 x 106 motile spermatozoa resulted in lower pregnancy rates compared to 150 and 300 x 106 motile spermatozoa doses (40.9% versus 65.2 and 70.0%, respectively) and pregnancy rate was not affected by extenders. Insemination of chilled motile spermatozoa stored in either Triladyl or Optixcell resulted in similar pregnancy rates, regardless of insemination dose, although an upward trend with increasing sperm number was apparent (Triladyl; 11.1% versus 21.1% and Optixcell; 5.9% versus 12.5%, for 300 x 106 and 500 x 106 groups, respectively; P > 0.05). No pregnancies were obtained with frozen thawed semen. In conclusion, this study demonstrated that the success of camel AI is highly dependent on sperm dose, location of semen deposition, timing of insemination and semen type. Further studies are required to determine the reason for the compromised fertility of preserved semen despite apparent high in vitro quality.

Effect of carbohydrates on lipid metabolism during porcine oocyte IVM.

Porcine oocytes contain a large amount of endogenous lipid, which is thought to function as an intracellular source of energy. The aim of this study was to determine the effects of stimulating or inhibiting lipid metabolism using l-carnitine or etomoxir respectively on the IVM of porcine oocytes cultured in media of varying carbohydrate composition. In the presence of pyruvate and lactate, exclusion of glucose inhibited oocyte nuclear and cytoplasmic maturation compared with oocytes matured in media containing low (1.5mM) and high (4.0mM) concentrations of glucose. In the absence of pyruvate and lactate in low-glucose medium only, a greater proportion of l-carnitine-treated oocytes progressed to the MII stage compared with untreated oocytes. The inclusion of pyruvate and lactate significantly altered the distribution of cytoplasmic lipid droplets and elevated the ATP content of oocytes, whereas the l-carnitine treatment did not. Further, the inhibitory effect of etomoxir on nuclear maturation was decreased in high- compared with low-glucose medium. The results indicate that carbohydrate substrates are absolutely necessary for effective porcine oocyte maturation, and that l-carnitine supplementation can only partially compensate for deficiencies in carbohydrate provision.

Effects of pre-freeze Nigella sativa oil supplementation on cryosurvival of ovine spermatozoa.

The study was designed with three experiments to evaluate the effects of pre-freeze supplementation of Nigella sativa oil (NSO) and thymoquinone (TQ) on total motility, progressive motility, biokinetic characteristics, acrosomal integrity and DNA integrity of cryopreserved ovine spermatozoa. Semen samples collected from three proven fertile Merino rams were diluted with a Tris-based cryomedia containing different levels of NSO (Experiment I: 0, 10, 100 and 1,000 g/ml), TQ (Experiment II: 0, 1, 10 and 20 g/ml) and their optimum levels (Experiment III: 100 g/ml of NSO, 10 g/ml of TQ and 1 mM of α-tocopherol and cryopreserved as pellet (200µL) and subsequently evaluated at different post-thaw incubation periods (0, 2 and 4 hr). The results revealed that the percentage of total motility, progressive motility and biokinetic characteristics such as average path velocity, curvilinear velocity and straight-line velocity were higher (p < 0.05) in the sperm aliquots cryopreserved with 100 g/ml NSO or 10 g/ml TQ than in the sperm aliquots cryopreserved without supplementation just after thawing and 2 hr of post-thaw incubation. Among the supplements, NSO (100 g/ml) showed higher values of the total motility, progressive motility, biokinetic characteristics specially, average path velocity, curvilinear velocity and straight-line velocity, acrosome integrity and DNA integrity compared with the spermatozoa frozen without supplementation. Therefore, the results suggest that NSO may be added to the cryomedium to improve the cryosurvival of ovine spermatozoa.

A Coding Variant in the Gene Bardet-Biedl Syndrome 4 (BBS4) Is Associated with a Novel Form of Canine Progressive Retinal Atrophy.

Progressive retinal atrophy is a common cause of blindness in the dog and affects >100 breeds. It is characterized by gradual vision loss that occurs due to the degeneration of photoreceptor cells in the retina. Similar to the human counterpart retinitis pigmentosa, the canine disorder is clinically and genetically heterogeneous and the underlying cause remains unknown for many cases. We use a positional candidate gene approach to identify putative variants in the Hungarian Puli breed using genotyping data of 14 family-based samples (CanineHD BeadChip array, Illumina) and whole-genome sequencing data of two proband and two parental samples (Illumina HiSeq 2000). A single nonsense SNP in exon 2 of BBS4 (c.58A > T, p.Lys20*) was identified following filtering of high quality variants. This allele is highly associated (PCHISQ = 3.425e-14, n = 103) and segregates perfectly with progressive retinal atrophy in the Hungarian Puli. In humans, BBS4 is known to cause Bardet-Biedl syndrome which includes a retinitis pigmentosa phenotype. From the observed coding change we expect that no functional BBS4 can be produced in the affected dogs. We identified canine phenotypes comparable with Bbs4-null mice including obesity and spermatozoa flagella defects. Knockout mice fail to form spermatozoa flagella. In the affected Hungarian Puli spermatozoa flagella are present, however a large proportion of sperm are morphologically abnormal and <5% are motile. This suggests that BBS4 contributes to flagella motility but not formation in the dog. Our results suggest a promising opportunity for studying Bardet-Biedl syndrome in a large animal model.

Adjusting cryodiluent composition for improved post-thaw quality of rabbit spermatozoa.

Improved fertility following artificial insemination with frozen-thawed spermatozoa would offer rabbit producers faster genetic improvement. Previous work investigating cryoprotectants for rabbit spermatozoa have reported inconsistent results. Semen was collected from three rabbit bucks by artificial vagina and frozen using a standard procedure with varied cryodiluent components. Post-thaw analysis encompassed motility, sperm kinematic parameters and acrosome and membrane integrity. Spermatozoa were evaluated at 0, 2 and 4 h after thawing. Experiment 1 compared diluents with 3.5% dimethyl sulfoxide (DMSO), 1.5% acetamide, 1.75% DMSO + 0.75% acetamide or 3.5% DMSO + 1.5% acetamide. The treatment that resulted in the highest post-thaw motility (P<0.001) and acrosome integrity (P<0.001) was DMSO alone. Experiment 2 compared 3.5, 7 and 10% DMSO in the cryodiluent. The best post-thaw sperm motility (P<0.001) and linearity (P = .002) was in 3.5% DMSO, while 10% DMSO afforded higher acrosome/membrane integrity at this last time point (P<0.05). Experiment 3 varied the cryodiluent to contain either 9 or 17% egg yolk or 9 or 17% low density lipoproteins extracted from whole egg yolk. The treatment with the best post-thaw result was 17% egg yolk (motility, P = 0.01; acrosome/membrane integrity, P<0.001). Experiment 4 compared different carbohydrates in the cryodiluent; 50 mM glucose (TCG), 25 mM glucose with 25 mM sucrose (TCGS low), or 50 mM glucose with 50 mM sucrose (TCGS high). When data were pooled across time points, TCG had significantly higher motility than TCGS high (P = 0.021), but was not different from TCGS low. However, TCG had fewer spermatozoa with intact acrosomes and membranes than both TCGS low and TCGS high (P = .002). Put together, these results indicate that the best cryodiluent for rabbit spermatozoa frozen under the conditions used in this paper is with 7% DMSO and 17% egg yolk in a base medium containing 25 mM glucose and 25 mM sucrose.

Cryotolerance of porcine blastocysts is improved by treating in vitro matured oocytes with L-carnitine prior to fertilization.

Sufficient generation of adenosine triphosphate (ATP) by oocytes is critical for fertilization and embryo development. The objective of this study was to determine the effects of supplementing media with L-carnitine, a co-factor required for the metabolism of fatty acids, during the peri-fertilization period on embryo development and energy generation. Firstly, in vitro matured (IVM) porcine oocytes were co-incubated with sperm in IVF medium supplemented with 0‒24 mM L-carnitine. The blastocyst formation rate of the control group was greater than those of the L-carnitine groups (P < 0.05), except for the 3 mM L-carnitine group. Subsequently, oocytes and/or sperm were treated without or with 3 mM L-carnitine for either the 1 h pre-IVF oocyte incubation; the pre-IVF sperm preparation; the first 30 min of IVF; or the entire 5.5 h of IVF. Despite similar fertilization rates among the groups, the cleavage rate of the pre-IVF oocyte group was significantly greater than those of the other groups, except for the pre-IVF sperm group. Additionally, the oocyte ATP content and the cryotolerance of the resulting blastocysts were examined following the pre-IVF oocyte treatment. Oocyte ATP content was also similar among the groups (P > 0.05). Following vitrification, the post-warming survival rate of blastocysts derived from L-carnitine-treated oocytes was greater than that of blastocysts derived from untreated oocytes (42.4% vs. 24.9%; P < 0.05). In conclusion, a 1 h oocyte exposure to 3 mM L-carnitine immediately prior to insemination enhanced cleavage and improved the cryotolerance of resulting blastocysts. While the findings are suggestive of a lipolytic action, further studies are required to clarify the contributions of lipid metabolism and oxidative mechanisms to the observed effects of the L-carnitine treatment.

Copper-zinc superoxide dismutase deficiency impairs sperm motility and in vivo fertility.

Oxidative stress, overproduction of reactive oxygen species (ROS) in relation to defence mechanisms, is considered to be a major cause of male infertility. For protection against the deleterious effects of ROS, animals have a variety of enzymatic antioxidants that reduce these molecules to less reactive forms. The physiological role of these antioxidants in vivo has been explored extensively through genetic inhibition of gene expression; surprisingly, many of these animals remain fertile in spite of increased oxidative stress. Copper-zinc superoxide dismutase-deficient (Sod1(-/-)) male mice are one such example for which in vivo fertility has been repeatedly reported as normal, although examination of fertility has consisted of simply pairing animals of the same strain and checking for litters. This is a fairly low criterion by which to assess fertility. Herein, we show that Sod1-deficient males have zero fertilisation success in sperm competition trials that pit them against wild-type males of an otherwise identical genetic background and are almost completely infertile when mated singly with females of a different genotype. We also show that various aspects of sperm motility and function are impaired in Sod1-deficient mice. Testing the breeding capabilities of mice under more ecologically relevant conditions and with females of different genotypes may help reveal additional physiological causes of infertility.

Cryopreservation of epididymal alpaca (Vicugna pacos) sperm: a comparison of citrate-, Tris- and lactose-based diluents and pellets and straws.

Epididymal spermatozoa were harvested from male alpacas and frozen after extension and cooling to 4 degrees C in citrate-, Tris- and lactose-based diluents (Experiment 1) and as pellets in 0.25- and 0.5-mL straws on either dry ice or over liquid nitrogen vapour (Experiment 2) to determine the effects diluents and packaging on their motility and acrosome integrity. In Experiment 1, sperm motility was higher after cooling to 4 degrees C and after freeze-thawing (0 but not 3 h post-thaw) for spermatozoa extended in the lactose- than the citrate- or Tris-based diluent (P < 0.05). Post-thaw acrosome integrity after cooling to 4 degrees C and post-thaw (0 h) was reduced for spermatozoa frozen in citrate- compared with lactose- or Tris-based diluents, but was similar for all groups 3 h after thawing. In Experiment 2, sperm motility immediately after thawing was higher for pellet freezing than for 0.25- or 0.5-mL straws on dry ice or liquid nitrogen vapour (P < 0.05), although by 3 h post-thaw motility was similar for pellets and straws (P > 0.05). Acrosome integrity was similar for all groups immediately after thawing and 3 h post-thaw. Cryopreservation of epididymal alpaca spermatozoa is feasible, with retained motility and acrosome integrity post-thaw. Freezing as pellets in a lactose-based diluent is recommended.

Ligand-activated signal transduction in the 2-cell embryo.

Platelet-activating factor (PAF) is an autocrine trophic/survival factor for the preimplantation embryo. PAF induced an increase in intracellular calcium concentration ([Ca2+]i) in the 2-cell embryo that had an absolute requirement for external calcium. L-type calcium channel blockers (diltiazem, verapamil, and nimodipine) significantly inhibited PAF-induced Ca2+ transients, but inhibitors of P/Q type (omega-agatoxin; omega-conotoxin MVIIC), N-type (omega-conotoxin GVIA), T-type (pimozide), and store-operated channels (SKF 96365 and econazole) did not block the transient. mRNA and protein for the alpha1-C subunit of L-type channels was expressed in the 2-cell embryo. The L-type calcium channel agonist (+/-) BAY K 8644 induced [Ca2+]i transients and, PAF and BAY K 8644 each caused mutual heterologous desensitization of each other's responses. Depolarization of the embryo (75 mM KCl) induced a [Ca2+]i transient that was inhibited by diltiazem and verapamil. Whole-cell patch-clamp measurements detected a voltage-gated channel (blocked by diltiazem, verapamil, and nifedipine) that was desensitized by prior responses of embryos to exogenous or embryo-derived PAF. Replacement of media Ca2+ with Mn2+ allowed Mn2+ influx to be observed directly; activation of a diltiazem-sensitive influx channel was an early response to PAF. The activation of a voltage-gated L-type calcium channel in the 2-cell embryo is required for normal signal transduction to an embryonic trophic factor.