Low-Fat, High-Carbohydrate Diets Reduce Body Weight and Sperm Count but Increase Sperm Motility in Mice.

BACKGROUND: Male reproduction is impacted by both over- and under-nutrition, demonstrated by animal studies using high-fat and low-protein dietary interventions. Little is known about the impacts of low-fat, high-carb diets and types of dietary carbohydrates on sperm traits. OBJECTIVES: Using a nutritional geometry approach, we investigated the effects of partially or completely substituting glucose for fructose in isocaloric diets containing either 10%, 20%, or 30% fat (by energy) on sperm traits in mice. METHODS: Male C57BL/6J mice were fed 1 of 15 experimental diets for 18 wk starting from 8 wk of age. Reproductive organs were then harvested, and sperm concentration, motility, and velocity were measured using Computer-Assisted Sperm Analysis. RESULTS: Increasing dietary fat from 10% to 30% while maintaining energy density at 14.3 kJ/g and protein content at 20% resulted in increased body weight and sperm production but reduced the percentage of motile sperm. Body weight and seminal vesicle weight were maximized on diets containing a 50:50 mix of fructose and glucose, but carbohydrate type had few significant impacts on epididymal sperm traits. CONCLUSIONS: The opposing impacts of dietary fat on mouse sperm quantity and quality observed suggest that male fertility may not be optimized by a single diet; rather, context-specific dietary guidelines targeted to specific concerns in semen quality may prove useful in treating male infertility.

Molecular insights to the sperm-cervix interaction and the consequences for cryopreserved sperm.

Cryopreserved ram spermatozoa are limited in their capacity to traverse the ovine cervix and achieve fertilization. This altered interaction may be related to modified molecular communication between frozen-thawed ram spermatozoa, seminal plasma, and the female tract. As such, this review aims to identify the biological processes which underpin sperm maturation and transport throughout the female reproductive tract to elucidate factors which may alter this natural process in cryopreserved ram spermatozoa. We also assess critical barriers to ram spermatozoa specific to the ovine cervix and the role of seminal plasma in mitigating these barriers. Transcriptomics is explored as a new approach to understand the sperm-cervix interaction. Recent studies have demonstrated that both spermatozoa and seminal plasma contain a complex profile of coding and non-coding RNAs. These molecular species have clear links with functional fertility, and mounting evidence suggests they may be altered by cryopreservation. Emerging in vitro cell culture models are also investigated as a "next step" in studying this interaction, utilizing transcriptomics to identify subtle changes in female tract gene expression in response to spermatozoa. The application of such models is proposed as an exciting opportunity to investigate the unique challenges faced by cryopreserved spermatozoa traversing the ovine cervix prior to fertilization.

HDL mediates reverse cholesterol transport from ram spermatozoa and induces hyperactivated motility.

Reverse cholesterol transport or cholesterol efflux is part of an extensive plasma membrane remodeling process in spermatozoa that is imperative for fertilization. For ram spermatozoa, sheep serum is well known to support in vitro fertilization (IVF), but knowledge of its explicit role is limited. Though, it is postulated to elicit cholesterol efflux owing to the presence of high-density lipoproteins (HDLs) that interact with transmembrane cholesterol transporters, such as adenosinetriphosphate (ATP)-binding cassette transporter A1 (ABCA1) and scavenger receptor class B, type I (SR-BI). In this study, we report that both sheep serum and HDLs were able to elicit cholesterol efflux alone by up to 20-40% (as measured by the boron dipyrromethene (BODIPY)-cholesterol assay). Furthermore, when the antagonists glibenclamide and valspodar were used to inhibit the function of ABCA1 and SR-BI or ABCA1 alone, respectively, cholesterol efflux was only marginally reduced (8-15%). Nevertheless, it is likely that in ram spermatozoa, a specific facilitated pathway of cholesterol efflux is involved in the interaction between cholesterol acceptors and transporters. Interestingly, exposure to HDLs also induced hyperactivated motility, another critical event required for successful fertilization. Taken together, this study details the first report of the dual action of HDLs on ram spermatozoa, providing both an insight into the intricacy of events leading up to fertilization in vivo as well as demonstrating the possible application of HDL supplementation in media for IVF.

Quantitative Proteomic Analysis of Seminal Plasma, Sperm Membrane Proteins, and Seminal Extracellular Vesicles Suggests Vesicular Mechanisms Aid in the Removal and Addition of Proteins to the Ram Sperm Membrane.

Quantitative proteomic studies are contributing greatly to the understanding of the spermatozoon through the provision of detailed information on the proteins spermatozoa acquire and shed in the acquisition of fertility. Extracellular vesicles (EVs) are thought to aid in the delivery of proteins to spermatozoa in the male reproductive tract. The aim of this study is to isolate, identify and quantify EV proteins isolated from ram seminal plasma. Ram sperm plasma membrane proteins are also isolated using nitrogen cavitation and identified to better understand the interplay of proteins between the sperm membrane and extracellular environment. The categorization of proteins enriched in the EV population according to their function revealed three main groupings: vesicle biogenesis, metabolism, and membrane adhesion and remodeling. The latter group contains many reproduction-specific proteins that show demonstrable links to sperm fertility. Many of these membrane-bound proteins show testicular expression and are shed from the sperm surface during epididymal maturation (e.g., testis expressed 101; TEX101 and lymphocyte Antigen 6 Family Member K; LY6K). Their association with seminal EVs suggests that EVs may not only deliver protein cargo to spermatozoa but also assist in the removal of proteins from the sperm membrane.

Synergism between albumin, bicarbonate and cAMP upregulation for cholesterol efflux from ram sperm.

Compared to other mammalian species, ram spermatozoa are difficult to capacitate in vitro. Dibutyryl cAMP (db-cAMP) and the phosphodiesterase (PDE) inhibitors, caffeine and theophylline (cAMP up-regulators), must be added to traditional capacitation media (containing bicarbonate, calcium and BSA) to elicit a capacitation response. In this exploratory study, we assessed whether bicarbonate was still required for ram spermatozoa if cAMP is up-regulated by the addition of db-cAMP and PDE inhibitors and what role BSA plays in cholesterol efflux under these conditions. In this study, the validated BODIPY-cholesterol assay was used for the first time in ram spermatozoa to quantify cholesterol efflux by tracking the loss of BODIPY-cholesterol from the sperm plasma membrane using flow cytometry. The results show that under cAMP up-regulated conditions, an increase in membrane fluidity and tyrosine phosphorylation of sperm proteins remain as bicarbonate-dependent processes. In fact, the supplementation of bicarbonate under these conditions was necessary to further enhance cAMP production in ram spermatozoa, which correlated with the presence of these capacitation-related processes. When BSA was supplemented with cAMP up-regulators (as well as bicarbonate), there was a loss of approximately 20-23% of BODIPY-cholesterol (79.5 ± 30.5% to 76.9 ± 12.3% remaining from 10 min), indicating that BSA is essential for mediating cholesterol efflux in ram spermatozoa as measured by the BODIPY-cholesterol assay. The current study identifies the functional relationship between bicarbonate, BSA and cAMP up-regulators that is required to support capacitation-related processes in ram spermatozoa, specifically cholesterol efflux.

Binder of Sperm Proteins 1 and 5 have contrasting effects on the capacitation of ram spermatozoa.

Binder of Sperm Proteins (BSPs) are the most abundant seminal plasma protein family in the ram and bull. They have been extensively studied in the bull but less is known about their function in ovine seminal plasma and current knowledge suggests that BSPs may have different effects in these two species. In the bull, they facilitate capacitation and destabilize the sperm membrane during in vitro handling, whereas in the ram, they appear to stabilize the sperm membrane and prevent cryopreservation-induced capacitation-like changes. Further investigation into the effects of BSPs on ram spermatozoa under capacitating conditions is required to further clarify their physiological roles in the ram. We investigated the effects of Binder of Sperm Proteins 1 and 5 on epididymal ram spermatozoa in conditions of low, moderate, and high cAMP. BSPs had minimal effects on sperm function in low-cAMP conditions, but caused significant changes under cAMP upregulation. BSP1 stabilized the membrane and qualitatively reduced protein tyrosine phosphorylation, but significantly increased cholesterol efflux and induced spontaneous acrosome reactions. BSP5 slightly increased spontaneous acrosome reactions and caused sperm necrosis. However, BSP5 had minimal effects on membrane lipid order and cholesterol efflux and did not inhibit protein tyrosine phosphorylation. These findings demonstrate that under maximal cAMP upregulation, BSP1 affected ram spermatozoa in a manner comparable to bull spermatozoa, while BSP5 did not.

Proteomes of the Female Genital Tract During the Oestrous Cycle.

The female genital tract includes several anatomical regions whose luminal fluids successively interact with gametes and embryos and are involved in the fertilisation and development processes. The luminal fluids from the inner cervix, the uterus and the oviduct were collected along the oestrous cycle at oestrus (Day 0 of the cycle) and during the luteal phase (Day 10) from adult cyclic ewes. The proteomes were assessed by GeLC-MS/MS and quantified by spectral counting. A set of 940 proteins were identified including 291 proteins differentially present along the cycle in one or several regions. The global analysis of the fluid proteomes revealed a general pattern of endocrine regulation of the tract, with the cervix and the oviduct showing an increased differential proteins abundance mainly at oestrus while the uterus showed an increased abundance mainly during the luteal phase. The proteins more abundant at oestrus included several families such as the heat shock proteins (HSP), the mucins, the complement cascade proteins and several redox enzymes. Other proteins known for their interaction with gametes such as oviductin (OVGP), osteopontin, HSPA8, and the spermadhesin AWN were also overexpressed at oestrus. The proteins more abundant during the luteal phase were associated with the immune system such as ceruloplasmin, lactoferrin, DMBT1, or PIGR, and also with tissue remodeling such as galectin 3 binding protein, alkaline phosphatase, CD9, or fibulin. Several proteins differentially abundant between estrus and the luteal phase, such as myosin 9 and fibronectin, were also validated by immunohistochemistry. The potential roles in sperm transit and uterine receptivity of the proteins differentially regulated along the cycle in the female genital tract are discussed.

Binder of Sperm Proteins protect ram spermatozoa from freeze-thaw damage.

Cryopreservation causes sub-lethal damage which limits the fertility of frozen thawed spermatozoa. Seminal plasma has been investigated as a cryoprotectant, but has yielded inconsistent results due to considerable variation in its constituents. Individual seminal plasma proteins offer an ideal alternative to whole seminal plasma, and several have been correlated with freezing success. Binder of Sperm Proteins (BSPs) are abundant ram seminal plasma proteins which have been suggested to have significant protective effects on ram spermatozoa during cold shock. This is in direct opposition to bull spermatozoa, where BSPs cause sperm deterioration during in vitro handling. We investigated the potential of BSP1 and BSP5 to prevent freezing associated damage to important functional parameters of ram spermatozoa. BSPs purified by size exclusion chromatography improved post thaw motility and penetration through artificial mucus. Highly purified BSP1 and BSP5, isolated by gelatin affinity and RP-HPLC, improved motility and membrane integrity, and reduced post thaw protein tyrosine phosphorylation. Exposure to BSP5 before freezing increased the amount of phosphatidylethanolamine on the sperm surface after thawing. Neither BSP1 nor BSP5 prevented freezing associated changes in membrane lipid disorder. These results suggest that BSPs may significantly improve freezing outcomes of ram spermatozoa.

Proteomic Investigation of Ram Spermatozoa and the Proteins Conferred by Seminal Plasma.

Sperm proteomes have emerged for several species; however, the extent of species similarity is unknown. Sheep are an important agricultural species for which a comprehensive sperm proteome has not been produced. In addition, potential proteomic factors from seminal plasma that may contribute to improved fertility after cervical insemination are yet to be explored. Here we use liquid chromatography-tandem mass spectrometry to investigate the proteome of ejaculated ram spermatozoa, with quantitative comparison to epididymal spermatozoa. We also present a comparison to published proteomes of five other species. We identified 685 proteins in ejaculated ram spermatozoa, with the most abundant proteins involved in metabolic pathways. Only 5% of ram sperm proteins were not detected in other species, which suggest highly conserved structures and pathways. Of the proteins present in both epididymal and ejaculated ram spermatozoa, 7% were more abundant in ejaculated spermatozoa. Only two membrane-bound proteins were detected solely in ejaculated sperm lysates: liver enriched gene 1 (LEG1/C6orf58) and epidermal growth factor-like repeats and discoidin I-like domains 3 (EDIL3). This is the first evidence that despite its relatively complex proteomic composition, seminal plasma exposure leads to few novel proteins binding tightly to the ram sperm plasma membrane.

The Assessment of Sperm DNA Integrity: Implications for Assisted Reproductive Technology Fertility Outcomes across Livestock Species.

Sperm DNA integrity is increasingly considered a useful measure of semen quality in mammalian reproduction. However, the definition of DNA integrity, the ideal means by which it should be measured, and its predictive value for fertility remain a topic of much discussion. With an emphasis on livestock species, this review discusses the assays that have been developed to measure DNA integrity as well as their correlation with in vitro and in vivo fertility.

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.

Motility Assessment of Ram Spermatozoa.

For successful fertilisation to occur, spermatozoa need to successfully migrate through the female reproductive tract and penetrate the oocyte. Predictably, poor sperm motility has been associated with low rates of fertilisation in many mammalian species, including the ram. As such, motility is one of the most important parameters used for in vitro evaluation of ram sperm quality and function. This review aims to outline the mechanical and energetic processes which underpin sperm motility, describe changes in motility which occur as a result of differences in sperm structure and the surrounding microenvironment, and assess the effectiveness of the various methods used to assess sperm motility in rams. Methods of subjective motility estimation are convenient, inexpensive methods widely used in the livestock industries, however, the subjective nature of these methods can make them unreliable. Computer-assisted sperm analysis (CASA) technology accurately and objectively measures sperm motility via two-dimensional tracing of sperm head motion, making it a popular method for sperm quality assurance in domesticated animal production laboratories. Newly developed methods of motility assessment including flagellar tracing, three-dimensional sperm tracing, in vivo motility assessment, and molecular assays which quantify motility-associated biomarkers, enable analysis of a new range of sperm motion parameters with the potential to reveal new mechanistic insights and improve ram semen assessment. Experimental application of these technologies is required to fully understand their potential to improve semen quality assessment and prediction of reproductive success in ovine artificial breeding programs.

Factors Affecting the Survival of Ram Spermatozoa during Liquid Storage and Options for Improvement.

Semen preservation is an essential component of reproductive technologies, as it promotes genetic gain and long-distance semen transport and multiplies the number of ewes able to be inseminated per single ejaculate. However, the reduced temperature during cold storage at 5 or 15 °C inflicts sub-lethal damage to spermatozoa, compromising sperm quality and the success of artificial breeding. New and emerging research in various species has reported the advantages of storing spermatozoa at higher temperatures, such as 23 °C; however, this topic has not been thoroughly investigated for ram spermatozoa. Despite the success of storing spermatozoa at 23 °C, sperm quality can be compromised by the damaging effects of lipid peroxidation, more commonly when metabolism is left unaltered during 23 °C storage. Additionally, given the biosafety concern surrounding the international transport of egg-yolk-containing extenders, further investigation is critical to assess the preservation ability of synthetic extenders and whether pro-survival factors could be supplemented to maximise sperm survival during storage at 23 °C.

Increasing the Yield and Cryosurvival of Spermatozoa from Rhinoceros Ejaculates Using the Enzyme Papain.

The preservation of rhinoceros semen is vital for captive breeding programs. While successful collection and cryopreservation of rhinoceros semen has been reported, the volume and quality of semen produced is often low due to the high viscosity associated with ejaculates collected via electroejaculation. Reducing semen viscosity would enable access to previously unusable spermatozoa from viscous fractions and could improve quality post-thaw. The enzyme papain successfully reduced the viscosity of camelid semen but has yet to be tested in wildlife species. This study assessed the influence of papain on the in vitro quality of rhinoceros spermatozoa during cryopreservation using advanced semen assessment. In experiment 1, the motility of spermatozoa from the viscous fraction of an ejaculate, either untreated or treated with papain and its inhibitor E-64 prior to cryopreservation, was assessed post-thaw. In experiment 2, spermatozoa from papain-treated viscous fractions were compared to spermatozoa frozen from untreated sperm-rich fractions pre-freeze, as well as after 0, 1.5 and 3 h of incubation post-thaw (37 °C). Papain significantly increased the quantity of spermatozoa collected from ejaculates, as well as the motility prior to freezing. Papain also improved the post-thaw motility, velocity, linearity and straightness of samples compared to sperm-rich samples, with no detriment to sperm viability, lipid membrane disorder, production of ROS or DNA integrity (p < 0.05). Results show the benefit of supplementing rhinoceros spermatozoa with papain prior to cryopreservation on sperm cryosurvival and demonstrates the potential of using papain to improve the success of cryopreservation protocols, not only for the rhinoceros, but also for other wildlife species.

Global Methylation and Protamine Deficiency in Ram Spermatozoa Correlate with Sperm Production and Quality but Are Not Influenced by Melatonin or Season.

This study assessed whether the seasonal effects of melatonin that upregulate ram reproductive function alter sperm global methylation or protamine deficiency and whether these parameters corresponded to ram endocrinology, semen production and quality. Ejaculates were assessed from rams that received melatonin implants (n = 9) or no implants (n = 9) during the non-breeding season. Ejaculates (n = 2/ram/week) were collected prior to implantation (week 0), 1, 6 and 12 weeks post implantation and during the following breeding season (week 30). Flow cytometry was used to assess the sperm global methylation and protamine deficiency in each ejaculate, which had known values for sperm concentration, motility, morphology, DNA fragmentation, seminal plasma levels of melatonin, anti-Mullerian hormone and inhibin A. Serum levels of testosterone and melatonin were also evaluated. Though there was no effect of melatonin or season, sperm protamine deficiency was negatively correlated with sperm production and seminal plasma levels of anti-Mullerian hormone and positively correlated with sperm DNA fragmentation and morphology. Global methylation of spermatozoa was positively correlated with sperm DNA fragmentation, morphology and serum testosterone and negatively correlated with sperm motility. These moderate associations with sperm production and quality suggest that sperm protamine deficiency and global methylation are indicative of ram testicular function.

From One Ejaculate to Another: Transference of Sperm Traits via Seminal Plasma Supplementation in the Ram.

Males can adjust sperm motility instantaneously in response to the perceived risk of sperm competition. The speed of this response suggests that sperm motility is regulated by changes in seminal plasma rather than changes in the sperm cells themselves. Hence, here we test whether inter-ejaculate variation in seminal plasma can be used to alter sperm quality prior to use in assisted reproductive technologies. We supplemented fresh ejaculates of Merino rams with seminal plasma collected from previous 'donor' ejaculates to test whether changes in sperm kinetics were related to the relative quality of donor to focal ejaculates. We found a positive relationship between the change in sperm traits before and after supplementation, and the difference in sperm traits between the donor and focal ejaculate. Hence, sperm motility can be either increased or decreased through the addition of seminal plasma from a superior or inferior ejaculate, respectively. This positive relationship held true even when seminal plasma was added from a previous ejaculate of the same ram, although the slope of the relationship depended on the identity of both the donor and receiver ram. These findings indicate that seminal plasma plays a key role in the control and regulation of sperm kinetics, and that sperm kinetic traits can be transferred from one ejaculate to another through seminal plasma supplementation.

Novel methods to detect capacitation-related changes in spermatozoa.

Prior to interaction with the oocyte, spermatozoa must undergo capacitation, which involves a series of physio-chemical transformations that occur in the female tract. As capacitation is a pre-requisite for successful fertilisation, it is a topic of great interest for sperm biologists, but the complexity of the numerous biochemical and biophysical processes involved make it difficult to measure. Capacitation is an extremely complex event that encompasses numerous integrated processes that can occur concurrently during this window of time. The identification of techniques to accurately assess and quantify capacitation is therefore crucial to gain a meaningful insight into this fascinating sperm maturation event. Whilst there are extensive reviews in the literature that focus on the functional changes to spermatozoa during capacitation, few have examined the methods required to measure these changes. The aim of this review is to highlight frequently used methods to quantify different stages of capacitation and identify promising novel techniques. Factors that are able to modulate various capacitation processes will also be discussed. The overall outcome is to provide researchers with a toolbox of methods that can be used to gain a deeper understanding of the intricacies of capacitation in spermatozoa.

Seminal plasma proteins as markers of sperm fertility.

During ejaculation and deposition in the female genital tract, spermatozoa are exposed to seminal plasma, a mix of secretions primarily from the accessory sex glands. Proteins, which make up the largest contribution to seminal plasma by weight, have been the focus of much interest, in particular the identification of specific proteins both in the plasma and/or found bound to the sperm surface post ejaculation. Global proteomic studies of seminal plasma originating from a range of species over the last 15 years have revealed their hitherto unknown diversity and complexity. Seminal plasma is generally known to aid sperm survival and fertility in a range of species and studies have begun to reveal its link with sperm function and identification, as markers of fertility. This review summarises recent data on proteins found on the sperm surface that originate from seminal plasma and have subsequently been shown to correlate with fertility, with a focus on the pig.

Review on Sperm Sorting Technologies and Sperm Properties toward New Separation Methods via the Interface of Biochemistry and Material Science.

Successful fertilization in mammals requires spermatozoa to efficiently traverse the female reproductive tract to meet the egg. This process naturally selects high quality sperm cells for fertilization, but when artificial reproductive technologies are used such as in vitro fertilization, intracytoplasmic sperm injection, or intrauterine insemination, other methods of sperm selection are required. Currently, technology enables sperm sorting based on motility, maturity as defined by zeta potential or hyaluronic acid binding site expression, absence of apoptotic factors, appropriate morphology, and even sex. This review summarizes current knowledge on all known methods of sperm cell sorting, compares their efficiency, and discusses the advantages and limitations of each technique. Scope for further refinement and improvement of current methods are discussed as is the potential to utilize a variety of materials to innovate new methods of sperm separation.

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.