Stability during storage of LC-PUFA-supplemented infant formula containing single cell oil or egg yolk

The medical community recommends that infant formulae should mimic human milk as far as possible, particularly in regards to long-chain polyunsaturated fatty acids (LC-PUFA). These include arachidonic acid (AA) and docosahexaenoic acid (DHA), both of which provide biochemical and functional benefits to neonates. However, LC-PUFAs are highly susceptible to oxidation and the composition of formulae must be carefully controlled. In this study, the stability of two types of LC-PUFA-supplemented milk-based powdered infant formula was evaluated over the course of 18 months storage at 25 °C and 40 °C. One contained egg yolk phospholipids (IF-EPL) and the other contained triacylglycerides (DHA and AA) synthesized by single cell oils (IF-SCO). The following parameters were monitored: peroxide values, volatile content (propanal, pentanal and hexanal), fatty acid profiles, and potential and free furfural content (5-hydroxymethyl-2-furaldehyde and 2-furaldehyde). In addition, these formulae were subjected to sensory evaluations by a panel of experts. The parameters studied revealed acceptable lipid stability in both types of formula, with better results for IF-EPL. At the end of the study period, significant deficits (p < 0.05) in linoleic acid were noted in both formulae. However, no significant decreases were observed in the other fatty acids, including AA and DHA. In regards to furfural content, both formulae exhibited a similar increase, indicative of the typical Maillard reaction characteristic of products stored for long periods.     

Analysis of vitamins A,E and C,iron and selenium contents in infant milk-based powdered formula during full shelf-life

The stability of vitamins A, E and C, and the iron and selenium content were determined in two types of long chain-polyunsaturated fatty acid (LC-PUFA) supplemented milk-based powdered infant formulas (IF), during an 18-month storage period at 25 and 40 °C. The first type (IF-A) was supplemented with vitamin A as retinol acetate. The second type (IF-B) was supplemented with vitamin A as retinol palmitate. Both types were also supplemented with vitamin E as α-tocopherol acetate and with vitamin C as ascorbic acid. The two formulas studied had higher vitamin A (140% and 139%), vitamin E (109% and 198%) and vitamin C (167% and 118%), but lower iron (65.0% and 65.3%) and selenium (72.9% and 79.4%) than the amounts declared on the label. As expected, all the studied vitamins showed decreases during storage, and these decreases were higher in formulas stored at 40 °C. The losses of vitamin A at 40 °C after 18 months of storage were 27.5% in IF-A and 29% in IF-B, while vitamin E losses under the same conditions were 23.1% and 28.1%, and vitamin C losses under the same conditions were 28.4% and 48.6%. All these losses justify the over-fortification of the aforementioned vitamins in these LC-PUFA supplemented IFs. Iron and selenium content remained unchanged throughout storage.     

Evolution of potential and free furfural compounds in milk-based infant formula during storage

HPLC-DAD was used to monitor the evolution of potential and free furfural compounds (5-hydroxymethyl-2-furaldehyde, HMF; 2-furaldehyde, F; 2-furyl methyl ketone, FMC; and 5-methyl-2-furaldehyde, MF) over the shelf life of two types of infant powder formula: one supplemented with long-chain polyunsaturated fatty acids (LC-PUFA) in the form of microencapsulated fish oil (MFO), the other not supplemented with LC-PUFA. Following production, the formulae were stored at 25 and 37 °C. The initial furfural content in the supplemented formula was as follows: potential HMF (485.88 μg/100 g), potential F (167.13 μg/100 g), and free HMF (58.23 μg/100 g), while free F was not detected. In the unsupplemented formula, the following values were recorded: potential HMF (515.85 μg/100 g), potential F (170.29 μg/100 g), free HMF (84.92 μg/100 g), and free F (1.19 μg/100 g). In general, increased furfural content was observed during storage, an increase more pronounced in formula stored at 37 °C. Hardly any differences in furfural evolution during storage were observed between supplemented and unsupplemented infant formulae. The results suggest that the uses of MFO material for supplement formula not affected furfural formation. Neither FMC nor MF was formed in the formulae studied.     

Characterisation and optimisation of physical and oxidative stability of structured lipid-based infant formula emulsion: Effects of emulsifiers and biopolymer thickeners

Response surface methodology (RSM) was applied to investigate the effects of lecithin (0–0.4 g/100 ml), monoacylglycerol (0–0.4 g/100 ml), locust bean gum (LBG; 0–0.1 g/100 ml), and carrageenan (0–0.02 g/100 ml) on the physical and oxidative properties of structured lipid-based infant formula emulsion containing dairy proteins, lactose, vitamins, minerals and other micronutrients. Particle size, optical stability, viscosity, relative content of docosahexaenoic acid, stearidonic acid and total oxidation value were assessed during 28-day storage. ANOVA results showed that the experimental data were satisfactorily fitted to second-order polynomial models by multiple linear regression. The contour plots illustrated that lecithin and monoacylglycerol played a dominant role in controlling the emulsion stability compared to LBG and carrageenan. Lecithin content significantly affected all the responses measured, particularly lipid oxidation. Increasing monoacylglycerol concentration led to an increase in particle size and emulsion viscosity. The optimal condition to achieve the highest stability was predicted to be 0.2, 0.4, 0.045, and 0.015 g/100 ml lecithin, monoacylglycerol, LBG and carrageenan, respectively. The verification data further demonstrated the suitability of the models explored by RSM. Overall, the findings obtained in this study have important implications for the successful incorporation of structured lipid into infant formula emulsion for better infant nutrition and health.     

Vitamins A and E content in infant milk-based powdered formulae after opening the packet

Vitamins A and E were determined by HPLC in 20 starting, milk-based powdered infant formulae from local markets. We traced the evolution of these compounds, once the packets had been opened, during 0, 30 and 70 days of storage at room temperature (≈25 °C; min. 23 °C, max. 25.5 °C). Immediately after opening the packets, vitamin A ranged from 0.55 to 0.94 mg RE/100 g (93.3–183 μg RE/100 kcal) and vitamin E from 6.58 to 27.8 mg α-TE/100 g (1.36–5.39 mg α-TE/100 kcal). All the samples had higher vitamins A and E contents than those declared on the label, vitamin A mean adequacy values: 134% ± 17, min. 98%, max. 162%, and vitamin E 185% ± 47, min. 101%, max. 286%, including values at 0, 30 and 70 days of storage.     

Pesticide content of infant formulae and weaning foods available in New Zealand

A survey of the pesticide content of 25 commercially available infant formulae and 30 weaning foods available in New Zealand was undertaken in 1996. It included a representative mixture of imported and New Zealand manufactured infant foods. Three different pesticide screening techniques were used: a high-sensitivity organochlorine screen was carried out on all infant formulae, while a multiresidue screen (organochlorine and organophosphorus pesticides, synthetic pyrethroids, carbamate pesticides, fungicides and herbicides), and a specific screen for dithiocarbamate fungicides were both carried out on all weaning foods and on soy-based infant formulae. All results are expressed on a ready-to-feed basis. Extremely low levels of residues of three organochlorine compounds (p,p'-DDE, p,p'-DDT and dieldrin) were detected in infant formulae samples. Residues of p,p'-DDE were found in seven of 20 milk-based infant formulae at concentrations ranging from 0.03 to 0.5 μg kg^-1. Residues of p,p'-DDT were found in one imported milk-based infant formula at 0.7 μg kg^-1, and dieldrin residues were detected in four of five soy-based infant formulae at concentrations ranging from 0.05 to 0.08 μg kg^-1. The multiresidue pesticide screen detected low levels of residues of two organophosphorus pesticides; azinphos-methyl in one soy-based infant formula at a level of 22 μg kg^-1 and pirimiphos-methyl in two cereal-based weaning foods at concentrations of 5 and 14 μg kg^-1. None of the other approximately 140 pesticides (including fungicides and herbicides) included in the multiresidue screen were detected in any weaning foods or soy-based infant formulae, at a detection limit of 10 μg kg^-1. No residues of dithiocarbamate fungicides were detected in any product analysed, at a detection limit of 100 μg kg^-1.     

The determination of vitamin D3 in bovine milk by liquid chromatography mass spectrometry

A renewed international interest in vitamin D status has revealed significant deficiencies in several populations, including Australia. Vitamin D exists in two forms, cholcalciferol (D₃) and ergocalciferol (D₂). The main source of vitamin D₃ is from exposure of 7-dehydrocholesterol present in the skin to UV irradiation. However, there is an absolute requirement for vitamin D through proper dietary intake if humans live in the absence of sunlight or exclusively indoors. Bovine milk is considered to be a good dietary source of vitamin D₃, even though the levels are quite low. This paper describes robust methods using liquid chromatography–linear ion trap mass spectrometry (LC–MSⁿ) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) to measure the levels of vitamin D₃ in fresh bovine milk (0.05 μg/100 ml), commercial (natural and fortified) milk samples (0.01–2 μg/100 ml) and a dairy based infant formula (8 μg/100 g), without the need for extensive clean-up procedures. The limits of quantification (LOQ) are 0.01 μg/100 ml and 0.02 μg/100 ml for LC–MSⁿ and LC–MS/MS, respectively. Recoveries of vitamin D₃ added to the samples prior to saponification were satisfactory (range 60–90%). 25-Hydroxyvitamin D₃ was not present in any of the samples analysed (LOQ = 0.01 μg/100 ml, recovery range 30–40%).     

我国市售婴儿配方乳粉油脂配料使用情况分析

目的:了解我国市售婴儿配方乳粉的油脂配料使用情况及脂肪酸提供情况,为提升婴儿配方乳粉的营养水平及制定产品相关标准提供参考。方法:多渠道收集婴儿配方乳粉标签信息,统计分析油脂配料的种类、组合、最高添加量构成比及标识含量,比较全脂乳产品与脱脂乳产品、牛乳基产品与羊乳基产品、高必需脂肪酸产品与全部产品间的差异。均数和率的比较分别采用t检验和卡方检验。结果:共纳入269个婴儿配方乳粉。配料表分析显示,85%的产品使用了4种及以上的油脂配料,葵花籽油和椰子油在全部产品中的添加率最高,分别为88%、76%。牛、羊乳基配方粉的油脂配料使用情况存在差异,牛乳基配方粉中脂肪、亚油酸及α-亚麻酸的标识含量略高于羊乳基配方粉(P<0.05)。脱脂乳配方粉中,棕榈油添加率为32%,显著高于全脂乳产品(P<0.05)。44例使用了棕榈油的产品中仅有4例强化了1,3-二油酸2-棕榈酸甘油三酯。结论:牛、羊乳基配方粉中的必需脂肪酸标识含量基本一致。现市售婴儿配方乳粉以多种油脂组合使用的方式,以尽可能模拟母乳脂肪酸模式,但有些油脂类原料使用的科学性还有待进一步研究。     

Mineral retention in three-week-old piglets fed goat and cow milk infant formulas

Goat milk and cow milk are commonly used in infant formula preparations and, as such, understanding the nutritional characteristics of infant formulas made from these milks is important. In this study, a goat milk infant formula was compared with an adapted (whey-enhanced) cow milk infant formula with respect to mineral absorption and deposition using the 3-wk-old piglet as a model for the 3-mo-old infant. Equal numbers of piglets (n = 8) were fed either the goat milk formula or the cow milk formula. The mineral composition of the prepared goat milk formula was higher than that of the prepared cow milk formula for most minerals, including calcium (75.1 vs. 56.7 mg/100 mL) but excluding iron, which was higher in the prepared cow milk formula (0.92 vs. 0.74 mg/100 mL). The amounts of calcium, phosphorus, and manganese absorbed by the piglets were significantly higher for the goat milk formula, whereas the amounts of zinc, iron, and magnesium absorbed were significantly higher for the cow milk formula. Apparent mineral absorption, relative to intake, was statistically higher in the cow milk formula for calcium and phosphorus, although the actual differences were very small (less than 1.3%). For copper, zinc, iron, and magnesium there was no significant difference between treatments in apparent mineral absorption, whereas for manganese, absorption was higher for the goat milk infant formula. The absolute mineral deposition was higher in piglets fed the goat milk formula for calcium, phosphorus, and manganese, whereas iron deposition was higher in the piglets fed cow milk formula. For all other minerals tested, there were no significant differences between treatments. The goat milk infant formula provided a pattern of mineral retention in the 3-wk-old piglet very similar to that of the adapted cow milk infant formula. The minor differences observed between the 2 appeared to be due to the different mineral contents of the 2 formulas.     

Microdetermination of phosphorus from infant formulas, casein and casein phosphopeptides

A spectrophotometric method, which has been proposed for the determination of phosphorus in biological fluids based on the formation of a phosphomolybdate complex, is adapted and validated for the determination of phosphorus in milk-based infant formulas, casein, casein phosphopeptides (CPPs) and the soluble fractions resulting from their gastrointestinal digestion, as well as in the fractions resulting from the ion-exchange high-performance liquid chromatography (IE-HPLC) of CE90CPP and in the soluble fraction of infant formula. The detection and quantification limits (1.1 and 3.6 mg P/100 g sample, respectively) are low enough for the purpose described. The linearity (from 0.1 to 8 g of phosphorus in the assay) is adequate. The precision of the method, expressed as relative standard deviation, is lower than 1%, and the accuracy checked by the analysis of SRM 1846: milk-based powdered infant formula is good. The quality of the method, together with the low cost and ease of use, makes it suitable for routine analysis.     

Rapid determination of cholesterol in milk containing emulsified foods

In this study, a rapid and easy sample preparation method that involved no-heating saponification and dSPE (dispersive solid phase extraction) clean-up was developed to determine the level of cholesterol in milk containing emulsified foods (infant formula, baby food, cheese). The developed method utilised high performance liquid chromatography with an ultraviolet detector (HPLC-UVD) as a separation instrument. The optimum extraction conditions were determined as 10 mL isopropyl alcohol with 8.0 g (NH₄)₂SO₄ per 1 g sample, and saponification was achieved using 25 mg KOH, 1.6 g NaCl and 100 mg of a silica based NH₂. Cholesterol levels determined using CRMs (NIST SRM 1849 and 1544) were in the range of the certificated value and the recovery test using spiked materials ranged from 94.34% to 102.34% with a RSD of 0.63–4.10%. This method enables the accurate determination of cholesterol with reduced sample preparation time.     

Short communication: Investigation of aflatoxin M1 levels in infant follow-on milks and infant formulas sold in the markets of Ankara,Turkey

Aflatoxins are fungal toxins known to be carcinogenic and are classified as food contaminants. This study was performed to investigate aflatoxin (AF) M₁ levels in baby foods sold in Ankara (Turkey) and to evaluate the obtained results according to the Turkish Food Codex (TFC). For this purpose, a total of 84 baby food samples (50 follow-on milks and 34 infant formulas) were obtained from different markets in Ankara and the presence of AFM₁ in the samples was analyzed by ELISA. In 32 (38.1%) of 84 infant food samples, the presence of AFM₁ was detected in concentrations ranging between 0.0055 and 0.0201 µg/kg. The mean level (±standard error) of AFM₁ was found to be 0.0089 ± 0.0006 µg/kg in positive infant follow-on milks. Aflatoxin M₁ was detected in only 1 infant formula sample (2.94%) at a concentration of 0.0061 µg/kg. The extrapolated levels of AFB₁ contamination in feedstuffs were calculated based on levels of AFM₁ in baby food samples. The data estimating AFB₁ contamination in dairy cattle feedstuff indicate that contamination may range from 0.3410 to 1.2580 µg/kg, with the mean level (±standard error) being 0.5499 ± 0.0385 µg/kg, which is lower than the level set by the TFC and European Union regulations (5 µg/kg). According to the obtained results, the levels of AFM₁ in analyzed samples were within the allowed limit (0.025 µg/kg) set in the TFC. Low levels of AFM₁ in infant follow-on milks and infant formula samples obtained during the study do not pose a health risk to infants.     

Volatile compounds and fatty acid profiles in commercial milk-based infant formulae by static headspace gas chromatography: Evolution after opening the packet

The evolution of the volatile compounds propanal, pentanal and hexanal, and fatty acid profiles were examined in 20 infant formula (IF) milk powders during storage at 25 °C for 70 days after their packaging was opened. Few changes were observed in the fatty acid content during storage, but significant losses were found in C18:2 n − 6 and C18:3 n − 3 for some formulae. All three volatiles increased during storage in all formulae, confirming oxidative stability decreases once packets were opened. Significant correlation (p < 0.05) was detected between hexanal content and oxidation of n − 6 PUFA, specifically C18:2 n − 6 losses, and between propanal content and oxidation of n − 3 PUFA, specifically from C18:3 n − 3 losses.     

Ferritin synthesis by Caco-2 cells as an indicator of iron bioavailability: Application to milk-based infant formulas

The bioavailability of iron from milk-based infant formulas was estimated by an in vitro system including enzymatic digestion, iron uptake by Caco-2 cells and ferritin determination via an enzymoimmunoassay (ELISA). Positive correlations (p < 0.01) were found between the Fe(II) added to Caco-2 cells and ferritin synthesis and between the amount of dialyzed iron added to the cell culture and ferritin synthesis. The comparison of the bioavailability of iron from different milk-based formulas showed that adapted formulas having the same composition but differing in the iron salts added yielded similar ferritin levels. The same happened with follow-up formulas differing only in the presence or absence of bifidobacterium added. However, significant differences in the amount of ferritin synthesized were recorded between the two analyzed toddler formulas. Such differences could be attributed firstly to the ascorbic acid content and perhaps also to the manufacturing process involved, because one formula was in liquid form while the other was powdered.     

Impact of feed supplementation with different omega-3 rich microalgae species on enrichment of eggs of laying hens

Four different omega-3 rich autotrophic microalgae, Phaeodactylum tricornutum, Nannochloropsis oculata, Isochrysis galbana and Chlorella fusca, were supplemented to the diet of laying hens in order to increase the level of omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) in egg yolk. The microalgae were supplemented in two doses: 125 mg and 250 mg extra n-3 PUFA per 100 g feed. Supplementing these microalgae resulted in increased but different n-3 LC-PUFA levels in egg yolk, mainly docosahexaenoic acid enrichment. Only supplementation of Chlorella gave rise to mainly α-linolenic acid enrichment. The highest efficiency of n-3 LC-PUFA enrichment was obtained by supplementation of Phaeodactylum and Isochrysis. Furthermore, yolk colour shifted from yellow to a more intense red colour with supplementation of Phaeodactylum, Nannochloropsis and Isochrysis, due to transfer of carotenoids from microalgae to eggs.     

Optimization of a chocolate-flavored, peanut-soy beverage using response surface methodology (RSM) as applied to consumer acceptability data

Optimization of a chocolate-flavored, peanut-soy beverage was done using response surface methodology (RSM). Twenty-eight beverage formulations were processed by mixing three basic ingredients: peanut (X₁=30.6 g/100 g-58.7 g/100 g), soy (X₂=28.3 g/100 g-43.5 g/100 g), and chocolate syrup (X₃=13.0 g/100 g-25.9 g/100 g). The proportions of these ingredients were obtained using a three component, constrained mixture design where the source of soy was either flour (SF) or protein isolate (SPI). Consumer acceptability was measured in terms of nine response variables by 41 consumers using a 9-point hedonic scale. Parameter estimates were determined by performing regression analysis with no intercept option. l-pseudo-components were introduced to get equivalent second degree models further used to generate contour plots. The regions of maximum consumer acceptability [hedonic rating ?5.0 since the control (commercial chocolate milk) ratings were 6.0-7.0] were identified and marked on these contour plots for each sensory response. Superimposition of contour plots corresponding to each response variable resulted in optimum regions having consumer acceptability ratings ?5.0. Optimum formulations were all the combinations of X₁: 34.1 g/100 g-45.5 g/100 g, X₂: 31.2 g/100 g-42.9 g/100 g, and X₃: 22.4 g/100 g-24.1 g/100 g for SF-based; and X₁: 35.8 g/100 g-47.6 g/100 g, X₂: 31.2 g/100 g-43.5 g/100 g, and X₃: 18.3 g/100 g-23.6 g/100 g for SPI-based beverage formulations.     

HPLC-UV determination of total vitamin C in a wide range of fortified food products

A HPLC method for the quantification of total ascorbic acid (AA) and isoacorbic acid (isoAA) in fortified food products, premixes and duomixes has been developed. The method is based on the acidic extraction of AA in the presence of reducing agent Tris [2-carboxyethyl] phosphine (TCEP), which maintained AA in its reduced form. The separation was performed on a C₁₈ column with a sodium acetate eluent (pH = 5.4) containing TCEP and decylamine as ion pairing agent. The limit of detection was estimated at 0.1 mg/100 g and the recoveries were between 93% to 105% when spiking various food products with different amounts of AA. The intra-assay coefficient of variation value was 4.6% (n = 8) for infant formula and 0.8% (n = 9) for the premixes. The relative standard deviation reproducibility values obtained by 9 different laboratories ranged between 2.0% and 8.0% (n = 10). Application of the method to the analysis of 25 fortified food products, different premixes and duomixes revealed similar results to those found by the AOAC official titrimetry method.     

Inulin blend as prebiotic and fat replacer in dairy desserts: optimization by response surface methodology

The purpose of this work was to optimize the formulation of a prebiotic dairy dessert with low fat content (<0.1 g/100 g) using a mixture of short- and long-chain inulin. Response surface methodology was applied to obtain the experimental design and data analysis. Nineteen formulations of dairy dessert were prepared, varying inulin concentration (3 to 9 g/100 g), sucrose concentration (4 to 16 g/100 g), and lemon flavor concentration (25 to 225 mg/kg). Sample acceptability evaluated by 100 consumers varied mainly in terms of inulin and sucrose concentrations and, to a lesser extent, of lemon flavor content. An interaction effect among inulin and sucrose concentration was also found. According to the model obtained, the formulation with 5.5 g/100 g inulin, 10 g/100 g sucrose and 60 mg/kg of lemon flavor was selected. Finally, this sample was compared sensorially with the regular fat content (2.8 g/100 g) sample previously optimized in terms of lemon flavor (146 mg/kg) and sucrose (11.4 g/100 g). No significant difference in acceptability was found between them but the low-fat sample with inulin possessed stronger lemon flavor and greater thickness and creaminess.     

Study of spray drying of the Aloe vera mucilage (Aloe vera barbadensis Miller) as a function of its rheological properties

Spray Drying (SD) was used to obtain Aloe vera powder from fresh plants. The powder was reconstituted in an aqueous medium and its rheological properties, particle size distribution (PSD), thermal properties (differential scanning calorimetry, DSC), and morphology (scanning electron microscopy, SEM) were evaluated in order to find an alternative to natural gum to be used in the food industry. Rheological measurements were conducted at 25 °C in aqueous concentrations of 3 g/100 mL and 6 g/100 mL. A 2³ factorial design was used with three central points to evaluate yield, efficiency and the rheological properties of reconstituted powders, results were compared with a liophilized (FD) sample of A. vera mucilage. Experimental results showed that the shear viscosity decreased with the increase of the inlet air temperature and the speed of atomization, and it increased with increasing feed flow in SD. Additionally, most powders obtained in all treatments have an average particle diameter of ∼10 μm with a modal distribution (PSD). The best conditions of SD in order to obtain a good thickening agent were: 150 °C inlet temperature, 1.5 L/h feed rate and atomization speed of 275,000 rpm, and with rheological properties very close to those of the FD sample.     

Evaluation of gonadotropin-releasing hormone hydrogen chloride at 3 doses with prostaglandin F2α for fixed-time artificial insemination in dairy cows

The objectives of the current study were to evaluate the efficacy and field safety of GnRH HCl administered at 3 doses in fixed-time artificial insemination (FTAI) programs (Ovsynch) in dairy cows. A common protocol was conducted at 6 commercial dairies. Between 188 and 195 cows were enrolled at each site (total enrolled = 1,142). Cows had body condition scores ≥2 and ≤4, were between 32 to 140 d in milk, and were clinically healthy. Within pen and enrollment day (enrollment cohort), cows were assigned randomly in blocks of 4 to each of 4 treatments: (1) 25 mg of PGF_2α on d 7 with FTAI 72 ± 2 h later (control); (2) 100 μg of GnRH on d 0, d 7 a dose of 25 mg of PGF_2α, and the second administration of 100 μg of GnRH (T100) administered either at 48 ± 2 h (d 9) after PGF_2α with FTAI 24 ± 2 h later or 56 ± 2 h (d 9) after PGF_2α and FTAI 17 ± 2 h later; (3) same as T100 with both injections of 150 μg of GnRH (T150); and (4) same as T100 with both injections of 200 μg of GnRH (T200). Three sites selected the first option and 3 sites selected the second option for the timing of the second injection of all doses of GnRH. Cows were observed daily for signs of estrus and adverse clinical signs. Cows not returning to estrus had pregnancy diagnosis between 42 and 65 d following FTAI. Pregnancies per FTAI (P/FTAI) were analyzed as a binary variable (1 = pregnant, 0 = not pregnant) using a generalized linear mixed model with a binomial error distribution and a logit link function. The statistical model included fixed effects for treatment, random effects of site, site by treatment, enrollment cohort within site, and residual. Parity (first vs. second or greater) was included as a covariate. For demonstration of effectiveness, α = 0.05 and a 2-tailed test were used. Fifty-two cows were removed from the study because of either deviation from the protocol, injury, illness, culling, or death. Among the remaining 1,090 cows, 33.9% were primiparous and 66.1% were multiparous. Back-transformed least squares means for P/FTAI were 17.1, 27.3, 29.1, and 32.2% for control, T100, T150 and T200, respectively. The P/FTAI for each GnRH dose differed from that of the control. No differences were detected in P/FTAI between GnRH doses. No treatment-related adverse events were observed. Mastitis was the most frequently observed adverse clinical sign, followed by lameness and pneumonia. This study documents the efficacy and safety of doses of 100 to 200 μg of GnRH as the HCl salt when used in Ovsynch programs.