The global rise in antimicrobial resistance (AMR) has intensified calls for responsible antimicrobial use in livestock farming, including the Australian pork industry. While stewardship efforts largely focus on husbandry and biosecurity, the routine inclusion of antimicrobials in chilled boar semen extenders used for artificial insemination remains relatively underexamined. Despite widespread use, international studies consistently report bacteriospermia in extended semen, raising concerns regarding the efficacy of current practices and their contribution to AMR development. Australia's closed-herd system provides a unique opportunity to investigate bacterial dynamics within a contained environment; however, the seminal microbiota and associated AMR profiles remain poorly characterised. This review synthesises evidence from peer-reviewed literature, industry reports, and international guidelines to critically examine the composition of boar seminal microbiota, sources of bacterial contamination, and environmental influences on bacterial load. It explores the impacts of bacteriospermia on sperm quality and fertility, and it critically evaluates the rationale for antimicrobial inclusion in semen extenders, including commonly used agents such as gentamicin and neomycin and, in some cases, critically important antimicrobials like ceftiofur and enrofloxacin. Emerging non-antibiotic alternatives, including natural antimicrobials, nanoparticles, and probiotics, are discussed in the context of their feasibility and potential role in reducing AMR risk.

The pork industry largely operates intensive practices which enables precise herd management through innovative monitoring procedures. However, these must first be evaluated to ensure they are effective and appropriate before implementation. In particular, reproductive status determination has ample scope for improvements as, despite the aid of a few technological advancements, it remains predominantly reliant upon observations, as it has been since the 1950’s. Three areas of pig reproduction were identified to have the largest potential for improvement: oestrus detection, pregnancy diagnosis, and parturition onset. Specifically, current oestrus detection methods rely on a set of behavioural cues that culminate in a standing reflex indicative of sexual receptivity and used by stockpersons to implement an insemination protocol. Similar behaviours, alongside real-time ultrasonography, are utilised for pregnancy detection, and parturition onset is estimated based on behavioural and physical signs of oncoming labour. Benefits of advanced monitoring include higher conception rates, reduced reproductive disorders, and increased piglet survival. The aim of this review was to summarise current monitoring methods of these reproductive states and evaluate minimally invasive novel tools within the literature. The majority of identified tools can be categorised as biomarkers or remote technology, for which limitations and barriers to industry uptake have been discussed. Despite advances in machine learning and rapid diagnostics, gaps remain in development of accurate monitoring tools that can be applied to various pig genotypes across their reproductive lifecycle. It is likely that a combination of approaches is key to commercial adoption.

In brief: Successful fertilization in pigs depends on sperm that can navigate the complex geometry of the sow's reproductive tract, yet current sperm preparation methods select only on motility and can damage cells. This study demonstrates that a biomimetic microfluidic device selects boar sperm on thigmotactic competence, yielding functionally superior populations and revealing species-specific differences in angular navigation. Abstract: Efficient sperm selection is crucial for reproductive success in pigs, where artificial insemination is the main method of assisted reproduction. Traditional methods such as density gradient centrifugation (DGC) can enrich motile sperm but may cause mechanical and oxidative stress and do not replicate the natural filters of the sow's reproductive tract. This study assessed a biomimetic microfluidic (MF) device, previously validated in cattle, sheep, and dogs, for enrichment of high-quality spermatozoa from chilled boar ejaculates (n = 18). Unprocessed extended semen (RAW), DGC, and MF samples were evaluated for concentration, computer-assisted sperm analysis kinematics, viability, morphology, acrosome integrity, DNA fragmentation (sperm chromatin structure assay), and thigmotactic navigation. Microfluidic processing yielded a lower concentration (5.73 ± 2.22 × 106/ml) than RAW (65.20 ± 23.33 × 106/ml) and DGC (22.68 ± 10.95 × 106/ml; both p < 0.0001), an ∼11-fold reduction consistent with functional enrichment. Microfluidic sperm showed higher total motility (91.0% vs 77.4% DGC, 67.1% RAW), progressive motility (74.8% vs 59.6% and 33.1%; 2.2-fold over RAW), intact acrosomes (81.4% vs 71.9% and 62.6%), and lower DNA fragmentation (0.77% vs 1.68% and 4.38%; ∼5.7-fold reduction versus RAW; all p < 0.0001). Angular microchannels revealed graded thigmotaxis, with navigation success decreasing from 95.8% at 45° to 87.9% at 90° and 69.4% at 135°, comparable to bull and dog but lower than ram (89.1% at 135°). Biomimetic microfluidics isolates functionally superior boar sperm, offering a pump-free alternative to centrifugation with potential to enhance artificial insemination and in vitro embryo production.

Cryopreservation of Asian elephant semen remains problematic with respect to maintenance of both adequate semen quality and fertility post-thaw. The present study compared the effects of the penetrating cryoprotectants glycerol and dimethyl sulfoxide (DMSO) in the freezing extender, in conjunction with the surfactant Equex STM paste, on Asian elephant spermatozoa during freezing and thawing. Eight ejaculates from three mature bulls were used for this study. Semen was diluted with Tris egg yolk-based extender which contained either glycerol or DMSO, either with or without Equex, and frozen over liquid nitrogen in straws. Sperm motility, membrane, acrosome, and DNA parameters were assessed over 2 h incubation post-thaw to evaluate the cryoprotective capabilities of each freezing extender. Spermatozoa frozen with glycerol resulted in higher total (P < 0.001) and progressive (P < 0.05) motility compared to spermatozoa diluted with DMSO, regardless of Equex inclusion in the freezing extender. When in the presence of Equex, Asian elephant spermatozoa demonstrated higher proportions of viability (P < 0.001) and intact acrosomes (P < 0.001) post-thaw, regardless of the type of cryoprotectant. A large variance between individual bulls was noticed when assessing sperm DNA integrity, nevertheless freezing extender with glycerol plus Equex showed the highest proportion of spermatozoa with intact DNA after 2 h post-thaw (P < 0.05). Altogether, these results indicate a Tris egg yolk-based freezing extender containing cryoprotectants glycerol and Equex should be integrated into current Asian elephant semen cryopreservation protocols to yield the best sperm quality post-thaw and thus improve fertilising potential.

Domestic dogs (Canis familiaris) generally reproduce non-seasonally. However, the Basenji is an exception with seasonal breeding cycles, although some Basenjis can successfully reproduce outside of the breed's usual breeding season. There has been little investigation into the inheritance of non-seasonal breeding in canids, and the results remain inconclusive. Our aim was to predict the mode of inheritance of non-seasonal breeding in Australian Basenjis under a monogenic model. We purchased Australian registration records, which include a pedigree of 5796 Basenjis. This allowed us to confirm the breed's usual breeding season in Australia, which was from May to July, although 12.5% of litters were born outside this time. Based on the timing of litters, we assigned dams as seasonal or non-seasonal breeders according to whether they produced any litters outside the breeding season. We predicted the mode of inheritance of non-seasonal breeding using specialised software which uses segregation analysis to predict the mode of inheritance of a trait with incomplete penetrance. The pedigree and segregation analysis suggest that non-seasonal breeding could be dominant with incomplete penetrance in Australian Basenjis. These findings should help increase the power of future work identifying genetic markers involved in the control of seasonal reproduction in canids.

Reproduction is one of the most important considerations for the livestock industry, presenting significant economic and animal health and welfare pressures for producers. Parturition, the process of giving birth, is known to be highly painful in many mammalian species, but the understanding of parturient pain in sows is limited. Farrowing, the process of parturition in pigs, is understudied compared to other livestock species, with very little research available specifically regarding pain. Pain can be detrimental to animal wellbeing; hence, it is vital for it to be reliably detected and managed in such a way that improves both sow and piglet health and welfare. Grimace scales have been developed as a method for pain detection and quantification in animals via observations of facial expression changes in response to painful stimuli. This presents a unique opportunity for improved pain assessment during farrowing, increasing the current understanding of farrowing dynamics and potentially enhancing farrowing management decisions to prioritise sow welfare. This review synthesises and critically analyses the current knowledge on sow parturient pain and the ability for the application of facial grimace scoring to measure pain severity. Grimace scoring was found to be an effective, simple and feasible method of pain assessment in a number of domestic species, and its recent application to farrowing is a promising development in the understanding and management of sow welfare during parturition.

The agricultural industries have embraced the use of technologies as they improve efficiency and food security. The pork industry is no exception to this, as monitoring techniques and artificial intelligence allow for unprecedented capacity to track the physiological and behavioural condition of individual animals. This article reviews a range of those technologies in reference to the detection of oestrus in sows, a time when the ability to precisely ascertain physiological and behavioural changes associated with fluctuating hormone levels can have an immense impact on the economic profitability of the farm. The strengths and weaknesses of each technique from a practical application perspective are discussed, followed by considerations for further research and refinement.

Protocols that enable prompt identification of sows in need of assistance during farrowing are important for optimal production outcomes. Change in the colour and appearance of the sclera of the eye can indicate increased stress. This warrants investigation into its use for the identification of sows in need of assistance at the time of parturition. To this end, a three-level ocular scoring system for the pig was devised and subsequently used in a preliminary investigation into the relationship between farrowing kinetics and visual changes in the sclera at farrowing. Data were collected and analysed from twenty randomly selected Large White × Landrace crossbred sows during farrowing. It was determined that sows with a severe ocular score were more likely to experience a prolonged farrowing duration (p = 0.013) and incur increased parturient straining of either total straining time (p = 0.011) or straining per piglet (p = 0.025). There was a significant association between ocular score and litter size (p = 0.043). Ocular score was not associated with sow parity (p = 0.728) and inter-piglet interval (p = 0.075). The proposed three-level scoring system successfully identified sows experiencing an abnormal parturition as defined by a prolonged farrowing duration and increased straining time. Findings from this study suggest the potential application of this simple ocular scoring tool for identifying sows experiencing an abnormal farrowing via in-person application or integration into remote monitoring systems in the future.

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.

Context. The removal or supplementation of ejaculates with seminal plasma (SP) can affect cryotolerance and post-thaw survival of spermatozoa in many species. In the Asian elephant (Elephas maximus), elucidation of the SP proteome and investigation of how it affects spermatozoa may enable improvement of cryopreservation protocols. Aims. Herein, we characterise the Asian elephant SP proteome and investigate the impacts of SP on sperm cryotolerance in the presence of conspecific or heterospecific SP. Methods. Proteomic analysis of Asian elephant SP was performed using mass spectrometry on nine samples from three individuals. In a separate study, SP was removed from six ejaculates and spermatozoa were resuspended in Tris extender supplemented with: no seminal plasma (NOSP), conspecific SP from ejaculates exhibiting 'good' (GSP, >60%) or mixed sperm total motility (MSP), or horse SP (HSP). Samples underwent cryopreservation, and sperm parameters were compared prior to cryopreservation and after thawing (0 and 2 h). Key results. Mass spectrometry identified 155 proteins from an array of families. Significant differences were observed in post-thaw sperm quality between SP treatments: high concentrations of MSP (25%, v/v) displayed greater average path and straight-line velocity immediately after thawing (P < 0.05) and greater sperm motility index and beat cross frequency than NOSP after 2 h post-thaw incubation (P < 0.05). The addition of HSP improved sperm kinematic parameters compared to NOSP and GSP treatments (P < 0.05). Conclusions and implications. These preliminary findings suggest the potential of SP to enhance the cryosurvival of Asian elephant spermatozoa, with HSP showing particularly promising results compared to conspecific SP (GSP). Further research into the specific effects of Asian elephant SP proteins is warranted.

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.

Abstract This chapter starts with a brief overview of the endocrine system, which largely controls male reproduction and spermatogenesis. This is followed by a description of each of the major structures that comprise the male reproductive tract. This chapter is rounded off with a discussion on the components of semen.

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.

This study compared protocols for cryopreservation of ejaculated, papain-treated alpaca spermatozoa. This included different concentrations of egg yolk (EY; 5, 10 or 15%) and glycerol (2, 5 or 10%), diluent types (SHOTOR, lactose, skim milk or INRA-96™), freeze rates (2, 4 or 8 cm above liquid nitrogen; LN), thaw rates (37 °C for 1 min or 42 °C for 20 sec) and storage vessels (pellets, 0.25 mL straws or 0.5 mL straws). Spermatozoa were assessed pre-freeze and 0, 30, 60 and 90 min post-thaw. Forty-one hembras were inseminated with either fresh, papain-treated or frozen-thawed spermatozoa. Motility was affected by EY concentration (P < 0.001), diluent type (P < 0.001), freeze rate (P = 0.003) and storage vessel (P = 0.001). Viability was affected by EY concentration (P < 0.001), diluent type (P < 0.001), storage vessel (P = 0.002) and thaw rate (P = 0.03). For artificial insemination (AI), semen was diluted 1:3 in a lactose-based diluent, with 5% EY and glycerol. Freezing was in 0.5 mL straws, 2 cm above LN for 4 min then thawing at 37 °C for 1 min. Pregnancy rates of those ovulated (n = 26) were not different (1/5 fresh, 1/4 papain-treated, 0/17 frozen-thawed; P = 0.10). Pregnancy can be achieved after AI with papain-treated spermatozoa. Further work is needed to determine the optimal dose, timing and location for insemination.

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.

This study was conducted to assess the effects of commercial extenders and storage temperature on dromedary camel sperm quality during liquid preservation. In Experiment 1, ejaculates (n = five males; replicated seven times) were split and diluted with synthetic (OPTIXcell, EquiPlus, INRA96, Bioxcell or AndroMed; Experiment la) or egg-yolk based (Biladyl, Green buffer or Triladyl; Experiment 1b) extenders and stored for 48 h at 4 degrees C. In Experiment 2, split ejaculates (n = five males; replicated six times) were used to directly compare Green buffer, OPTIXcell and Triladyl extenders over 48 h of storage at 4 degrees C. Ejaculates collected in Experiment 3 (n = five males; replicated five times) were diluted with Green buffer or Triladyl before chilled storage for 48 h at 4 or 15 degrees C. Sperm kinematics, viability and acrosome integrity were assessed during liquid storage. In Experiment la, there was the greatest total sperm motility (TM) in the OPTIXcell group following 24 and 48 h of storage, while in Experiment 1b, there was the greatest TM after 48 h of storage with Triladyl and Green buffer. In Experiment 2, there were greater TM and viable acrosome intact spermatozoa in the Triladyl and Green buffer than with OPTIXcell group. In Experiment 3, there was a greater TM in the Triladyl than Green buffer group at 24 and 48 h of storage regardless of storage temperature (which had no effect on sperm quality). In conclusion, camel sperm have greater viability when preserved in liquid form for 48 h following dilution with Triladyl and storage at either 4 or 15 degrees C.

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.

The effect of semen collection frequency (once or twice per week) on the sexual behaviour, libido and semen characteristics (volume, colour, gross activity, viscosity, sperm concentration, morphology, motion characteristics and membrane viability and acrosome integrity) of dromedary camels (n = 7) was investigated over the course of 8 weeks. Results showed that frequency of collection influenced male camel libido (P < 0.05) but not sexual behaviour. Once per week collection frequency resulted in greater gross activity (2.7 ± 0.1 compared with 1.7 ± 0.1, P < 0.001) and greater sperm concentration (403 ± 16 compared with 261 ± 18 × 106 spermatozoa/mL, P < 0.001) compared to ejaculates collected twice per week. When collected twice per week, ejaculates collected during the first 3 weeks had a greater sperm concentration than those collected from week 4 onwards (P < 0.001). All ejaculates (100%) collected once per week ‘qualified’ (Criteria: > 60% total motility or > 100 × 106 spermatozoa/mL) for subsequent processing, but when collected twice per week the percentage of qualified ejaculates dropped sharply after three weeks (P < 0.001; 69% of ejaculates qualified over 8- week collection period). Twice weekly collection frequency caused a reduction (P < 0.001) in progressive motility, path velocity, track speed, lateral head amplitude, beat cross frequency and straightness. In conclusion, during the peak breeding season, semen can be collected twice per week from dromedary male camels for a period of 3 weeks only or once per week for 8 weeks without affecting semen quality.

ContentsThe study was designed with three experiments to evaluate the effects of pre‐freeze supplementation ofNigella sativaoil (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 a pellet (200 µL) 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 especially, 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.